Systems and methods for measuring cell signaling protein activity - Patents.com

Abstract

Described herein are systems for measuring enzyme activity. Described herein are methods for measuring or screening enzyme activity utilizing the systems described herein.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 144,766, filed February 2, 2021, the entirety of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on January 24, 2021 has the filename 52652-708_601_SL.txt and is 56,814 bytes in size. [Background technology]

[0003] Measuring cell signaling pathway responses is one of the cornerstones of modern medicine. Cell signaling pathway responses may result from the innate activity of the cell. Cell signaling pathway responses can be stimulated by an exogenous source, such as by contacting a cell with a ligand that binds to a receptor expressed on the cell's surface. Contact between the ligand and the cell surface receptor initiates changes in enzyme activity that comprise a signaling cascade within the cell, leading to changes in the cell signaling pathway response. Thus, measuring or screening enzyme activity can capture changes in the cell signaling pathway response initiated by a ligand contacting a cell surface receptor. Summary of the Invention

[0004] Described herein are systems and methods for interrogating and measuring cell signaling pathway responses, screening for antagonists, inverse agonists, or agonists of cell signaling pathways, or discovering new cell signaling pathways. Current methods utilize endogenous response element regulated promoters proximal to the nucleic acid encoding the reporter molecule. These methods suffer from a high degree of background signal of the reporter molecule due to the "leaky" nature of the endogenous response element binding promoter of the cell. These methods also suffer from a high coefficient of variation, false positives, or false negatives of measurable activity in the cell signaling pathway. Finally, such methods suffer from a low absolute value of reporter activation, resulting in a low ratio of measurable cell signaling activity to background.

[0005] In one embodiment, the system and method described herein comprises at least one nucleic acid encoding a regulatory element and / or effector of a cell signaling pathway. In some cases, the regulatory element upregulates or downregulates the expression of the effector. In some cases, the regulatory element upregulates or downregulates the expression of an exogenous effector. In some cases, the regulatory element upregulates or downregulates the expression of an endogenous effector. By adjusting the expression of the effector, the system and method increase the measurable response of the cell signaling pathway, increase the ratio of the measurable response of the cell signaling pathway to background, and decrease the coefficient of variation, false positives, or false negatives of the measurable cell signaling pathway.

[0006] In some embodiments, the present invention describes a system for measuring G protein-coupled receptor (GPCR) activity in mammalian cells, the system comprising a first effector of GPCR activity whose expression is upregulated compared to the expression of the first effector of GPCR activity in a wild-type state, and a second effector of GPCR activity whose expression is downregulated compared to the expression of the second effector of GPCR activity in a wild-type state. In some embodiments, the GPCR activity comprises Giα subunit activity, Gsα subunit activity, Gqα subunit activity, or G12 / 13α subunit activity. In some embodiments, the GPCR activity comprises Giα subunit activity. In some embodiments, the Giα subunit activity comprises Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, or Gαz activity. In some embodiments, the GPCR activity comprises Gsα subunit activity. In some embodiments, the GPCR activity comprises Gqα subunit activity. In some embodiments, the GPCR activity comprises a G12 / 13 α subunit activity. In some embodiments, the first effector of the GPCR activity comprises a first adenylyl cyclase. In some embodiments, the first adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10. In some embodiments, the first adenylyl cyclase is ADCY6. In some embodiments, the first adenylyl cyclase is encoded by a nucleic acid operably linked to a promoter or enhancer that overexpresses the first adenylyl cyclase compared to a wild-type state. In some embodiments, the second effector of the GPCR activity comprises a second adenylyl cyclase. In some embodiments, the second adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10. In some embodiments, the second adenylyl cyclase is ADCY3.In some embodiments, the second adenylyl cyclase is targeted by one or more of microRNA, shRNA, siRNA, CRISPR / Cas9 complex that reduces the expression of the second adenylyl cyclase compared to a wild-type state. In some embodiments, the system includes a reporter nucleic acid, which generates a detectable signal from a reporter gene that is proportional to the GPCR activity. In some embodiments, the reporter nucleic acid includes a cAMP response element (CRE) sequence operably linked to the reporter gene. In some embodiments, the reporter gene includes a unique molecular identifier (UMI) nucleic acid sequence. In some embodiments, the reporter gene includes one or more of expression of a unique molecular identifier, a fluorescent signal, a luminescent signal. In some aspects, the mammalian cell is selected from the group consisting of HEK293 cells, CHO-K1 cells, COS-7 cells, and U2OS cells. In some embodiments, the system is integrated into the genome of the mammalian cell.

[0007] In some aspects, methods are described herein for measuring GPCR activity in cells, comprising contacting a GPCR ligand with the system described herein, wherein the ligand complexes with the GPCR and initiates or inhibits GPCR activity. In some embodiments, the GPCR ligand is selected from the group consisting of a polypeptide and a small molecule.

[0008] In one aspect, the present specification describes a system for measuring G protein-coupled receptor (GPCR) activity, the system comprising: (a) a first effector of GPCR activity, whose expression is upregulated compared to the expression of the first effector in a wild-type state; and (b) a second effector of GPCR activity, whose expression is downregulated compared to the expression of the second effector in a wild-type state. In certain embodiments, the expression of the first effector is upregulated by contacting the GPCR with a GPCR activator. In certain embodiments, the GPCR activator is a GPCR agonist. In certain embodiments, the GPCR activator is an agonist of the first effector. In certain embodiments, the system comprises a first nucleic acid encoding the first effector of GPCR activity, which is operably linked to a regulatory element to upregulate the expression of the first effector. In certain embodiments, the expression of the second effector is downregulated by contacting the GPCR with a GPCR inhibitor. In certain embodiments, the GPCR inhibitor is a GPCR inverse agonist. In certain embodiments, the GPCR inhibitor is a GPCR antagonist. In certain embodiments, the GPCR inhibitor is an inverse agonist of the second effector. In certain embodiments, the GPCR inhibitor is an antagonist of the second effector. In certain embodiments, the system includes a second nucleic acid that downregulates expression of the second effector. In certain embodiments, the GPCR activity is a GPCR inhibitor that downregulates expression of the second effector. i α subunit activity, G s α subunit activity, G q α subunit activity, or G 12 / 13 In certain embodiments, GPCR activity includes α subunit activity. i In certain embodiments, the G i The α subunit activity includes activity of Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, or Gαz. In certain embodiments, the GPCR activity includes activity of Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, or Gαz. s In certain embodiments, GPCR activity includes α subunit activity. qIn certain embodiments, GPCR activity includes α subunit activity. 12 / 13The α subunit activity is included. In certain embodiments, the regulatory element is inducible. In certain embodiments, the regulatory element is constitutively active. In certain embodiments, the first effector comprises a first adenylyl cyclase. In certain embodiments, the first adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10. In certain embodiments, the first adenylyl cyclase is ADCY6. In certain embodiments, the second effector comprises a second adenylyl cyclase. In certain embodiments, the second adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10. In certain embodiments, the second adenylyl cyclase is ADCY3. In certain embodiments, the second nucleic acid comprises a microRNA, shRNA, siRNA, gRNA, or a combination thereof. In certain embodiments, the second nucleic acid comprises a microRNA. In certain embodiments, the second nucleic acid comprises a gRNA. In certain embodiments, the system further comprises a CRISPR-Cas system operably linked to the gRNA. In certain embodiments, the GPCR activity comprises cyclic AMP (cAMP) activity. In certain embodiments, the GPCR activity comprises a change in cAMP concentration. In certain embodiments, the GPCR activity comprises reverse cAMP activity. In certain embodiments, the system increases cAMP activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to cAMP activity in cells not comprising the system. In certain embodiments, the system increases the cAMP concentration in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to the cAMP concentration in cells not comprising the system.In certain embodiments, the system increases reverse cAMP activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to reverse cAMP activity in cells not comprising the system. iThe system further comprises a second regulatory element for upregulating the expression of the α subunit. In certain embodiments, the system comprises at least one additional effector. In certain embodiments, the at least one additional effector is adenylyl cyclase. In certain embodiments, the system further comprises a second regulatory element for upregulating the expression of at least one additional effector. In certain embodiments, the system increases the ratio between GPCR activity and background in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold, compared to the GPCR activity to background ratio in cells not comprising the system. In certain embodiments, the system increases the GPCR activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold, compared to the GPCR activity in cells not comprising the system. In certain embodiments, the system reduces the background in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to the background in cells not comprising the system. In certain embodiments, the system comprises a reporter nucleic acid, wherein the reporter nucleic acid generates a detectable signal proportional to GPCR activity. In certain embodiments, the reporter nucleic acid comprises a cAMP response element (CRE) sequence. In certain embodiments, the reporter nucleic acid is operably linked to a unique molecular identifier (UMI) nucleic acid sequence. In certain embodiments, the detectable signal comprises a fluorescent signal. In certain embodiments, the detectable signal comprises a luminescent signal. In certain embodiments, the first nucleic acid comprises a gene expression cassette for upregulating expression of a first effector. In certain embodiments, the second nucleic acid comprises a gene expression cassette for downregulating expression of a second effector. Also described herein are cells comprising the system described. In certain embodiments, the cell comprises a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is of mammalian origin.In certain embodiments, the cell is from a cell line. In certain embodiments, the cell is selected from the group consisting of CHO-K1 cells, COS-7 cells, and U2OS cells. In certain embodiments, the system is integrated into the genome of the cell. In certain embodiments, the cell comprises a cell population. In certain embodiments, the cell population comprises a population of eukaryotic cells. In certain embodiments, the cell population comprises a population of mammalian cells. In certain embodiments, the cell population comprises a population of cells derived from mammalian cells. In certain embodiments, the cell population comprises a population of cells derived from a cell line. In certain embodiments, the cell population comprises a population of cells selected from the group consisting of CHO-K1 cells, COS-7 cells, and U2OS cells. Also described herein is a method for measuring GPCR activity, the method comprising measuring GPCR activity in a cell or cell population. In certain embodiments, the GPCR activity is a steady-state GPCR activity. In certain embodiments, the cell or cell population is not contacted with a GPCR ligand. Also described herein is a method for measuring GPCR activity in cells, comprising contacting a cell or a cell population with a GPCR ligand, wherein the ligand forms a complex with GPCR and initiates or inhibits GPCR activity.In certain embodiments, the GPCR ligand is expressed in the cell.In certain embodiments, the GPCR ligand is selected from the group consisting of a polypeptide, a non-peptide compound, and a small molecule. [Brief description of the drawings]

[0009] This application for patent contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] 1 shows an exemplary embodiment described herein, in which the activity of a G-protein coupled receptor (GPCR) can be measured more accurately and robustly by up-regulating or down-regulating the expression of at least one family member of adenylyl cyclase (AC). Up-regulation or down-regulation of adenylyl cyclase is indicated by a double arrow. [Figure 2A] The design of the gene expression cassette described herein for increasing GPCR activity by altering the expression level of adenylyl cyclase (AC) is shown. Figure 2A shows an exemplary second generation gene expression cassette that can increase AC6 expression while knocking down AC3 expression. [Figure 2B] The design of the gene expression cassette described herein is shown to increase GPCR activity by changing the expression level of adenylyl cyclase (AC). Figure 2B shows an exemplary third generation gene expression cassette that can knock down AC3 expression by targeting AC3 with multiple guide RNAs to downregulate AC3 with transcriptional repressors. [Figure 2C] The design of the gene expression cassette described herein for increasing GPCR activity by altering the expression level of adenylyl cyclase (AC) is shown. Figure 2C shows an exemplary first generation gene expression cassette that increases only AC6 expression. [Diagram 3] We show that altering AC expression (MADSv2.5 cassette, e.g., second generation gene expression cassette in FIG. 2A) increased GPCR activity. [Figure 4] Luciferase measurements generated from cells containing the first generation gene expression cassette in FIG. 2C are shown, demonstrating that AC6 overexpression alone increased reverse CRE activity. [Diagram 5] Figure 2B shows platform data generated from cells containing a third generation gene expression cassette (including AC3 knockdown), thereby demonstrating an improvement over platform data generated from cells containing a second generation gene expression cassette as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used in the practice of the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0011] The use of absolute terms or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "firstly," "next," "then," "before," "after," "lastly," and "finally" are not intended to limit the scope of the embodiments disclosed herein, but are exemplary.

[0012] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0013] As used herein, the terms "at least one," "one or more," and "and / or" are broadly interpreted expressions that are conjunctive and discriminative in operation. For example, the terms "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" mean A alone, B alone, C alone, A and B, A and C, B and C, or A, B, and C, respectively.

[0014] As used herein, "or" may refer to "and," "or," or "and / or," and may be used both exclusively and inclusively. For example, the term "A or B" may refer to "A or B," "A but not B," "A but not B," and "A and B." In some cases, the context may dictate a particular meaning.

[0015] All of the systems and methods described herein are modular and not limited to sequential steps, and thus terms such as "first" and "second" do not necessarily imply a priority, critical order, or order of operation.

[0016] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, such as the limits of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation for a value, as is customary. In some cases, "about" or "approximately" refers to an amount in the vicinity of 10% (plus or minus) of the stated amount.

[0017] As used herein, the phrase "small molecule" refers to any molecule with a low molecular weight (e.g., 900 Daltons). In some cases, the molecule may be an organic compound that can regulate a biological process or reaction. In some cases, the small molecule can bind to a GPCR or a ligand of a GPCR to increase or decrease GPCR activity. In some cases, the small molecule is a ligand for a GPCR. Non-limiting examples of small molecules can include alfuzosin, terazosin, clonidine, bisoprolol, betaxolol, metoprolol, atenolol, albuterol, nadolol, penbutolol, tolterodine, atropine, scopolamine, calcimer, metoclopramide, haloperidol, olanzapine, ropinirole, pramipexole, loratadine, cetirizine, dimenhydrinate, cimetidine, ranitidine, trazodone, sumatriptan, exenatide, fentanyl, codeine, meperidine, oxycodone, montelukast, or misoprostol.

[0018] Where a specific value is described in this application and in the claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for that particular value.

[0019] The terms "increased", "increasing" or "increase" are used herein to generally mean an increase of a statistically significant amount. In some cases, the term "increased" or "increase" means an increase of at least 10% compared to a baseline value, such as at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, including a 100% increase, or any increase between 10-100% compared to a baseline value, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a baseline value.

[0020] The terms "decreased," "decreasing," or "decrease" are used herein to generally mean a statistically significant amount of decrease. In some cases, the terms "decreased" or "decrease" mean at least a 10% decrease compared to a baseline value, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% decrease (e.g., nonexistent or undetectable values ​​compared to a baseline value), including a 100% decrease, or anywhere between 10-100%. In the context of a marker or condition, these terms mean a statistically significant decrease in such a value. This decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, and is preferably decreased to a value accepted as within the normal range for individuals without the given disease.

[0021] As used herein, "cell" generally refers to a biological cell. A cell is a structural, functional, and / or biological unit of a living organism. A cell may be derived from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants, fungal cells (e.g., cells from yeast, mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), or cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, the cells are not derived from a natural organism (e.g., the cells may be synthetic and referred to as artificial cells). In some cases, the cells are primary cells. Optionally, the cells are from a cell line.

[0022] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides include synthetic nucleotides. Nucleotides include synthetic nucleotide analogs. Nucleotides are monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules that contain them. The term nucleotide as used herein may also refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

[0023] The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably to refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs, in either single-stranded, double-stranded, or multi-stranded form. In some cases, the polynucleotide is exogenous (e.g., heterologous polynucleotide). In some cases, the polynucleotide is endogenously present in a cell. In some cases, the polynucleotide may be present in a cell-free environment. In some cases, the polynucleotide is a gene or a fragment thereof. In some cases, the polynucleotide is DNA. In some cases, the polynucleotide is RNA. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. In some cases, the polynucleotide contains one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queousine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), guide RNA (gRNA), microRNA (miRNA), non-coding RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In some cases, the sequence of nucleotides is interrupted by non-nucleotide components.

[0024] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the polypeptide chains presented and other peptides, e.g., linkers and connecting peptides, may contain amino acid residues, including natural amino acid residues and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. In some embodiments, the polypeptide may contain modifications to the native or natural sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the protein or by errors due to PCR amplification. In some cases, the polypeptide encodes a gene or transgene as described herein.

[0025] As used herein, the term "gene" or "transgene" refers to a nucleic acid segment (also referred to as a "coding sequence" or "coding region") that codes for an individual protein or RNA, optionally with associated regulatory elements, such as a promoter, operator, terminator, etc., located upstream or downstream of the coding sequence. In some cases, the promoter is an inducible promoter. In some embodiments, the regulatory element includes at least one open reading frame (ORF) that does not code for a transgene. Instead, the ORF of the regulatory element can upregulate the transgene. In some embodiments, the ORF of the regulatory element can downregulate the transgene. In some embodiments, the ORF of the regulatory element is located 5' upstream of the transgene. In some embodiments, the ORF of the regulatory element is located 3' downstream of the transgene. The term "gene" or "transgene" should be interpreted broadly and may encompass mRNA, cDNA, cRNA, and genomic DNA forms of a gene. In some applications, the term "gene" encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'UTR), exons, and introns. In some genes, the transcribed region will include an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" or "transgene" includes only the coding sequences (e.g., "open reading frame" or "coding sequence") necessary to encode a polypeptide. In some embodiments, a gene or transgene does not encode a polypeptide, including, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some embodiments, the term "gene" or "transgene" includes not only the transcribed sequence, but also the non-transcribed region, including upstream and downstream control elements, including regulatory regions, enhancers, and promoters. The term "gene" or "transgene" may encompass mRNA, cDNA, and genomic forms of a gene.

[0026] The term "reverse cyclic AMP activity" refers to a decrease in cAMP activity or a decrease in cAMP concentration.

[0027] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and encoded polypeptide may collectively be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression may include a biological activity of a polypeptide encoded by a polynucleotide described herein. With respect to expression, "upregulation" generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the level of expression in the wild-type state. For example, expression of a gene or transgene may be upregulated by a system described herein by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more, as compared to expression of the gene or transgene in a wild-type state (e.g., when expression of the gene or transgene is not upregulated by a system described herein). "Downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence as compared to expression in a wild-type state. For example, expression of a gene or transgene may be downregulated by a system described herein by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more, as compared to expression of the gene or transgene in a wild-type state (e.g., when expression of the gene or transgene is not upregulated by a system described herein). "Wild-type" or "wild-type state" may refer to a phenotypic or biological measurement or observation of expression that occurs naturally, without manipulation with an expression vector or nucleic acid that reduces expression of the target (e.g., expression as the product of a normal allele as opposed to expression as the product of a mutant or engineered gene, or expression by an siRNA or CRISPR / Cas9 system).

[0028] Percent sequence identity (%) with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to obtain the maximum sequence identity percentage, and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various known means, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences to be compared.

[0029] The terms "identity", "identical", or "percent identical" as used herein to describe a nucleic acid sequence compared to a reference sequence can be determined using the formula set forth in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence identity can be determined using the most recent version of BLAST as of the filing date of this application.

[0030] The polypeptides of the systems described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transcribe a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genomic integration vector or "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid that can replicate extrachromosomally. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In some cases, the vector contains regulatory elements, such as promoters, enhancers, polyadenylation signals, and the like, for use in controlling transcription. The regulatory elements may be derived from mammalian, bacterial, viral, or insect genes. In embodiments, the vector contains a gene expression cassette as described herein. Typically, the ability to replicate in a host, conferred by an origin of replication, and a selection gene for facilitating recognition of transformants can be further incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may also be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors may contain sequences directing site-specific integration (e.g., AttP-AttB recombination) into a defined location or set of restricted sites within the genome. Additionally, vectors may contain sequences derived from transposable elements for integration.

[0031] As used herein, the term "transfection" or "transfected" refers to a method of intentionally introducing exogenous nucleic acid into a cell through a process commonly used in laboratory experiments. Transfection can be accomplished, for example, by lipofection, calcium phosphate precipitation, viral transduction, or electroporation. Transfection can be either transient or stable.

[0032] As used herein, the term "transfection efficiency" refers to the degree or proportion of a cell population that takes up an exogenous nucleic acid. Transfection efficiency can be measured as the percentage (%) of a given cell population that takes up an exogenous nucleic acid compared to the total cell population of the system. Transfection efficiency can be measured in both transiently or stably transfected cells.

[0033] As used herein, the term "bioactive polypeptide" refers to a polypeptide expressed by a cell that modulates the expression of a gene. A bioactive polypeptide can directly modulate the expression of a gene by signal transduction through one or more intermediary molecules or polypeptides, in response to a stimulus, or by any other mechanism. A bioactive polypeptide can be a transmembrane polypeptide (such as a receptor or channel protein), an intracellular polypeptide (a signal transduction intermediary), an extracellular polypeptide, or a secreted polypeptide.

[0034] As used herein, "reporter activity" refers to the empirical readout of a reporter. For example, a luciferase reporter will have a luminescent readout when incubated with an appropriate substrate. Other reporters, such as fluorescent proteins, may not require a substrate, but can be measured, for example, by a microscope or a fluorescent plate reader. In some embodiments, the reporter is encoded by a reporter nucleic acid. In some cases, the expression of the reporter is driven by a regulatory element or promoter as described herein. In some embodiments, the expression of the reporter is driven by a cAMP response element, such as cAMP response element binding protein (CREB).

[0035] overview The systems and methods described herein can measure GPCR activity by detecting the presence or absence and / or activation level of a response element-bound promoter associated with a GPCR activator. Also described herein are systems and methods for increasing GPCR activity. In some cases, the systems and methods described herein allow for GPCR activity with reduced background when measuring GPCR activity compared to conventional reporter systems and methods for measuring GPCR activity. In some cases, the systems and methods described herein allow for GPCR activity with increased levels of cAMP signal compared to conventional reporter systems and methods for measuring GPCR activity. In some embodiments, the systems and methods described herein include at least one nucleic acid encoding an effector described herein. In some cases, the at least one nucleic acid includes a regulatory element. In certain embodiments, the response element-bound promoter is activated upon termination of GPCR activity (e.g., the signal transduction cascade of the GPCR). In certain embodiments, the presence of the response element-bound promoter can be measured before and after an external stimulus, such as a peptide, macromolecule, compound, or small molecule that binds or complexes with the GPCR. In certain embodiments, the system or method is useful for screening for pharmaceutical discovery purposes. In some cases, the system or method includes at least one nucleic acid that includes a response element-regulated promoter that directs the expression of the reporter.In certain embodiments, the reporter is a polypeptide.In certain embodiments, the reporter may further include a unique molecular identifier (UMI), such as a unique nucleic acid sequence that can be used to identify a specific cell type or a specific cell population.

[0036] As a non-limiting example, the bioactive polypeptide may include a specific G protein-coupled receptor (GPCR) among hundreds known. Thus, the UMI element can easily and quickly collate the signaling of several different bioactive polypeptides in a multiplex format. Furthermore, the systems and methods provided herein reduce background by the response element regulated promoter. This allows for more accurate quantification and reduces the number of false positive test compounds in any multiplexed screen for compounds that may activate the bioactive polypeptide. In certain embodiments, the nucleic acid sequence encoding the reporter polypeptide is absent. In certain embodiments, the nucleic acid sequence encoding the UMI is absent. In certain embodiments, the nucleic acid sequence encoding the UMI is 5' of the nucleic acid sequence encoding the reporter polypeptide. In certain embodiments, the nucleic acid sequence encoding the reporter polypeptide is 5' of the nucleic acid sequence encoding the UMI.

[0037] In certain embodiments, the reporter-encoding nucleic acid encodes a reporter polypeptide. In certain embodiments, the reporter polypeptide is directly detectable. In certain embodiments, the reporter polypeptide generates a detectable signal upon the enzymatic activity of the protein on the substrate. In certain embodiments, the detection of the reporter polypeptide can be quantitatively achieved. In certain embodiments, the reporter polypeptide comprises a luciferase protein, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, secreted placental alkaline phosphatase, or a combination thereof. In certain embodiments, the reporter polypeptide is a luciferase protein, and non-limiting examples of substrates include firefly luciferin, rathiacin luciferin, bacterial luciferin, coelenterazine, dinoflagellate luciferin, vargurin, and 3-hydroxyhispidin.

[0038] In certain embodiments, the nucleic acid encoding the reporter may also encode UMI.UMI comprises a short sequence of nucleotides that is unique to nucleic acid.UMI may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides or more in length.UMI can be detected by any suitable method that can sequence UMI, such as next-generation sequencing.The detection method of UMI can be quantitative, including next-generation sequencing.

[0039] In certain embodiments, the present specification describes a method of using the system described herein for use in drug discovery, the system comprises at least one nucleic acid encoding an effector described herein and a reporter nucleic acid.In certain embodiments, the method comprises contacting a cell or a cell population with a nucleic acid under conditions sufficient for the nucleic acid to be internalized and expressed by the cell (e.g., transfected), contacting the cell with a physical or chemical stimulus, and determining the activation of the reporter by one or more assays.In certain embodiments, the method comprises contacting a cell or a cell population with a nucleic acid encoding a transcription factor nucleic acid and a reporter nucleic acid, and determining the activation of the reporter by one or more assays.

[0040] A non-limiting embodiment of the present disclosure is shown in FIG. 1. A cell (100) comprises a system for measuring GPCR activity as described herein. A GPCR (101) is contacted with a GPCR ligand (102). Upon contact between the GPCR (101) and the GPCR ligand (102), the Gα subunit of the G protein (103) dissociates from Gp and Gy and interacts with various members of the adenylyl cyclase (AC) family (104). By up-regulating or down-regulating the expression of various AC family members (104), GPCR activity can be measured more robustly and accurately by detecting the presence or absence and degree of expression of a reporter construct (105) as described herein. In some cases, the reporter construct (105) may optionally further comprise a nucleic acid sequence of a unique molecular identifier (UMI) that can be used to identify a particular cell type or a particular cell population, including the system described herein.

[0041] GPCR activity In some embodiments, systems and methods are described herein for measuring GPCR activity in cells. In some cases, the system includes a first nucleic acid that encodes a first effector of GPCR activity. In some cases, the first nucleic acid may be operably linked to a regulatory element that is configured to overexpress the first effector of GPCR activity. In some embodiments, the regulatory element includes a promoter, an enhancer, or a combination thereof. In some cases, the system may further include a second nucleic acid that reduces the expression of a second effector of GPCR activity. In some cases, the GPCR activity includes activity resulting from a ligand that binds to or complexes with a GPCR expressed by the cell. In some embodiments, the system includes an additional effector.

[0042] The GPCR may be any GPCR that produces GPCR activity. In some embodiments, the GPCR may be exogenous or endogenous to the cell comprising the system described herein. For example, the GPCR may be an exogenous GPCR that is introduced as a transgene into the cell comprising the system described herein. In some cases, the GPCR may be endogenous to the cell having the system described herein. In some cases, the GPCR may be stimulated by a ligand to produce GPCR activity. In some embodiments, the GPCR may produce GPCR activity independent of or in the absence of a ligand that is complexed or bound to the GPCR. In some embodiments, the GPCR activity includes an orphan GPCR activity, where the GPCR activity may result from a putative or unknown ligand that is complexed or bound to the GPCR.Exemplary GPCRs include adenosine receptors, beta-adrenergic receptors, neurotensin receptors, muscarinic acid receptors, 5-hydroxytryptamine receptors, adrenoceptors, anaphylatoxin receptors, angiotensin receptors, apelin receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, cholecystokinin receptors, dopamine receptors, endothelin receptors, free fatty acid receptors, bile acid receptors, galanin receptors, motilin receptors, ghrelin receptors, glycoprotein hormone receptors, GnRH receptors, histamine receptors, KiSS1-derived peptide receptors, leukotriene and lipoxin receptors, lysophospholipid receptors, melanin concentrating hormone receptors, melanocortin receptors, melatonin receptors, neuromedin U receptors, neuropeptide receptors, N -formyl peptide family receptors, nicotinic acid receptors, opioid receptors, opsin-like receptors, orexin receptors, P2Y receptors, peptide P518 receptors, platelet activating factor receptors, prokineticin receptors, prolactin releasing peptide receptors, prostanoid receptors, protease activated receptors, relaxin receptors, somatostatin receptors, SPC / LPC receptors, tachykinin receptors, trace amino receptors, thyrotropin releasing hormone receptors, urotensin receptors, vasopressin / oxytocin receptors, orphan GPCRs, calcitonin receptors, corticotropin releasing factor receptors, glucagon receptors, parathyroid receptors, VIP / PACAP receptors, LNB7™ receptors, GABA receptors, metabotropic glutamate receptors, or calcium sensor receptors.

[0043] In some embodiments, the ligands complexed or bound to the GPCR described herein are known. In some cases, the ligands for the GPCR may be agonists or partial agonists, and can complex or bind to the GPCR to increase GPCR activity in cells. In some embodiments, the ligands may be inverse agonists, where an inverse agonist is one in which the ligand binds or complexes to the GPCR to reduce basal GPCR activity. In some embodiments, the ligands complexed or bound to the GPCR may be antagonists, where an antagonist is one in which the ligand binds to the GPCR to block binding to the GPCR agonist. In some cases, the ligands are small organic or inorganic moieties. In some cases, the ligands may be polypeptides. The polypeptides may be naturally occurring GPCR binding proteins or other proteins that can bind or complex to the GPCR, or derivatives or fragments thereof. In some embodiments, the polypeptides may be antibodies that bind to the GPCR. The antibody may be any immunoglobulin, including any immunoglobulin isotype, subclass, monoclonal antibody, and fragments thereof that contain an antigen-binding domain, such as Fab, F(ab')2, single chain Fv (scFv), Fv, domain antibody (dAb), nanobody, and diabody. In some embodiments, the ligand may be covalently bound to the GPCR. In some embodiments, the ligand may be a peptidomimetic, a nucleic acid, a peptide nucleic acid (PNA), a polymer, or an aptamer. In some embodiments, the ligand may be an ion, such as Na+ or Zn2+. In some embodiments, the ligand may be a lipid. In some embodiments, the ligand may be a carbohydrate. In some embodiments, the ligand may be a ligand with a dissociation constant (K) of millimolar (mM) to picomolar (pM). d ) that can bind to GPCRs.

[0044] In some embodiments, the GPCR activity includes a G protein activity. In some cases, the G protein activity may be a stimulatory activity. In some cases, the G protein activity may be an inhibitory activity. G protein activity can include the activity of a G protein alpha subunit (Gα), a G protein beta subunit (Gβ), a G protein gamma subunit (Gγ), and / or a G protein beta gamma subunit (Gβγ). In some cases, the G protein activity includes the activity of a Gα. Gα activity can include the activity of a G i α subunit, G s α subunit activity, G q α subunit activity, or G 12 In some cases, the G protein activity may include the activity of the α subunit. i Contains α subunit activity. G i The α subunit is Gα i1 , Gα i2 , Gα i3 , Gα o , Gα t , Gα g , or Gα z G s The α subunit is Gα s or Gα oif G q The α subunit is Gα q , Gα 11 , Gα 14 , or Gα 16 The G12α subunit may comprise Gα 12 or Gα 13 In some cases, the Gβ subunits may include Gβ1, Gβ2, Gβ3, Gβ4, or Gβ5. In some cases, the Gγ subunits may include Gγ1, Gγ2, Gγ3, Gγ4, Gγ5, Gγ6, Gγ7, Gγ8, Gγ9, Gγ 10 , Gγ 11 , or Gγ 12 may include:

[0045] In some cases, the GPCR activity includes the activity of at least one effector of the GPCR. In some cases, the effector of the GPCR includes a second messenger associated with a G protein. For example, the GPCR activity includes an active effector, such as an adenylyl cycle, a phospholipase, a phosphodiesterase, a kinase, or an ion channel, which is a second messenger associated with a G protein activity. In some embodiments, the GPCR activity includes a cyclic AMP (cAMP) activity. In some embodiments, the GPCR activity includes a reverse cAMP activity. In some cases, the GPCR activity includes downstream signaling of a messenger or molecule associated with a G protein. For example, the GPCR activity can include the transcriptional or translational activity of a cAMP response element that binds to a reporter nucleic acid described herein. In some cases, the GPCR activity can be measured from a detectable signal generated from a cAMP response element that binds to a reporter nucleic acid described herein. In some embodiments, the GPCR activity is proportional to a detectable signal generated by a reporter described herein.

[0046] In some embodiments, the system described herein comprises at least one nucleic acid that regulates expression of any one of the effectors described herein. In some embodiments, the nucleic acid encodes any one of the effectors described herein. In some embodiments, the nucleic acid encodes a regulatory element that regulates expression of the effector. In some embodiments, the regulatory element comprises a promoter, an enhancer, or a combination thereof. In some embodiments, the regulatory element comprises a promoter. In some embodiments, the regulatory element comprises an enhancer. In some embodiments, the regulatory element upregulates expression of the effector. In some embodiments, the regulatory element downregulates expression of the effector. In some embodiments, the nucleic acid encodes both a regulatory element and an effector, where the regulatory element upregulates expression of the effector encoded by the nucleic acid. In some embodiments, the nucleic acid encodes both a regulatory element and an effector, where the regulatory element downregulates expression of the effector encoded by the nucleic acid. In some embodiments, the at least one nucleic acid encodes any one of the effectors described herein and any one of the regulatory elements described herein. In some embodiments, at least one nucleic acid encodes an effector and a regulatory element. For example, at least one nucleic acid can encode an effector operably linked to a regulatory element. In some embodiments, the system described herein comprises at least a first effector comprising a first adenylyl cyclase and at least a second effector comprising a second adenylyl cyclase. In some embodiments, the first effector and the second effector are under the expression control of different regulatory elements. In some embodiments, the first effector is encoded by a nucleic acid operably linked to a promoter or enhancer, where the promoter or enhancer increases or overexpresses the expression of the first effector compared to a wild-type state.In some embodiments, the first adenylyl cyclase is encoded by a nucleic acid operably linked to a promoter or enhancer, where the promoter or enhancer increases or overexpresses expression of the first adenylyl cyclase compared to a wild-type state.

[0047] In some embodiments, the effector regulated by the regulatory element is adenylyl cyclase. In some embodiments, the adenylyl cyclase is ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10. In some embodiments, the regulatory element upregulates any one of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10. In some embodiments, the regulatory element upregulates ADCY6. In some embodiments, the regulatory element downregulates any one of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10. In some embodiments, the nucleic acid encodes an RNA capable of downregulating the expression of any one of the effectors described herein. In some embodiments, the RNA comprises a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a guide RNA (gRNA), or a microRNA (miRNA) that downregulates the expression of the effector. In some cases, the gRNA can assist in complexing with a gRNA-guided nuclease and guide the nuclease to digest the nucleic acid of the effector.Non-limiting examples of gRNA-guided nucleases that can assist in digesting the effector nucleic acid (and subsequently reducing expression) include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases; The gRNA-guided nuclease may include a zirconium fusion nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR-associated RNA-binding protein, a recombinase, a flippase, a transposase, an Argonaute (Ago) protein (e.g., a prokaryotic Argonaute (pAgo), an archaeal Argonaute (aAgo), and an eukaryotic Argonaute (eAgo)), any derivatives thereof, any mutants thereof, and any fragments thereof. In some embodiments, the gRNA-guided nuclease may be a CRISPR-associated (Cas) protein. In some embodiments, the gRNA-guided nuclease may be a CRISPR / Cas9 protein. In some embodiments, the gRNA-guided nuclease, such as a CRISPR / Cas9 protein, may be encoded from the same nucleic acid that also encodes the gRNA. In some embodiments, the gRNA-guided nuclease, such as a CRISPR / Cas9 protein, may be encoded from a different nucleic acid that encodes the gRNA.

[0048] In some embodiments, the RNA comprises a microRNA that downregulates the expression of an effector. In some embodiments, the RNA comprises an siRNA, shRNA, gRNA, or miRNA that downregulates any one of the adenylyl cyclases described herein (e.g., ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10). In some embodiments, the RNA comprises an siRNA, shRNA, miRNA, or gRNA that downregulates ADCY3.

[0049] In some embodiments, at least one effector can be downregulated by knocking down or knocking out the activity of at least one effector in a cell by contacting the cell with an inhibitor. In some cases, the inhibitor can be an inhibitor of a GPCR. In some embodiments, the inhibitor can be an inhibitor of adenylyl cyclase. In some embodiments, the inhibitor can be an antagonist. In some embodiments, the antagonist can be an antagonist of a GPCR. In some embodiments, the antagonist can be an antagonist of adenylyl cyclase. In some embodiments, the inhibitor can be an inverse agonist. In some embodiments, the antagonist can be an inverse agonist of adenylyl cyclase. In some embodiments, the inhibitor can be an inverse agonist of adenylyl cyclase. Exemplary inhibitors of adenylyl agonists can include KH7, MDL12330A, NKY80, SKF83566, SQ22536, and ST034307.

[0050] In some embodiments, the system described herein comprises at least one effector that is upregulated by a first regulatory element and at least one other different effector that is downregulated by a second regulatory element. In some embodiments, the system comprises at least one adenylyl cyclase that is upregulated by a first regulatory element and at least one other different adenylyl cyclase that is downregulated by a second regulatory element. In some embodiments, the system comprises ADCY6 that is upregulated by a first regulatory element and ADCY3 that is downregulated by a second regulatory element.

[0051] In some embodiments, at least one effector can be upregulated in a cell by contacting the cell with an activator. In some cases, the activator can be an activator for GPCR. In some embodiments, the activator can be an activator for adenylyl cyclase. In some embodiments, the activator can be an agonist. In some embodiments, the agonist can be an agonist for GPCR. In some embodiments, the agonist can be an agonist for adenylyl cyclase. Exemplary activators of adenylyl agonists can include forskolin, NKH477, PACAP1-27, and PACAP1-38.

[0052] In some embodiments, the systems described herein increase GPCR activity. In some embodiments, the systems described herein increase measurable GPCR activity. In some embodiments, the measurable GPCR activity comprises measurable G protein activity. In some embodiments, the measurable GPCR activity comprises G protein alpha subunit (Gα) activity, G protein beta subunit (Gβ) activity, G protein gamma subunit (Gγ) activity, and / or G protein beta gamma subunit (Gβγ) activity. In some embodiments, the G protein comprises Gα activity. Gα activity comprises G i α subunit, G sα subunit activity, G q α subunit activity, or G 12 In some cases, the G protein activity may include the activity of the α subunit. i In some embodiments, the system comprises α subunit activity in cells that do not contain the system. i Compared with α subunit activity, G iIn some embodiments, the system increases measurable Giα subunit activity in a cell by at least about 0.1-fold to about 10,000-fold. In some embodiments, the system increases measurable Giα subunit activity in a cell by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5-fold, about 0.5 to about 10 times, about 0.5 to about 20 times, about 0.5 to about 50 times, about 0.5 to about 100 times, about 0.5 to about 500 times, about 0.5 to about 1,000 times, about 0.5 to about 10,000 times, about 1 to about 2 times, about 1 to about 5 times, about 1 to about 10 times, about 1 to about 20 times, about 1 to about 50 times, about 1 to about 100 times, about 1 to about 500 times, about 1 to about 1,000 times, about 1 to about 10,000 times, about 2 to about 5 times, about 2 to about 10 times, about 2 to about 20 times times, about 2 times to about 50 times, about 2 times to about 100 times, about 2 times to about 500 times, about 2 times to about 1,000 times, about 2 times to about 10,000 times, about 5 times to about 10 times, about 5 times to about 20 times, about 5 times to about 50 times, about 5 times to about 100 times, about 5 times to about 500 times, about 5 times to about 1,000 times, about 5 times to about 10,000 times, about 10 times to about 20 times, about 10 times to about 50 times, about 10 times to about 100 times, about 10 times to about 500 times, about 10 times to about 1,000 times, about 10 times to about 10,000 times, about from about 20-fold to about 50-fold, from about 20-fold to about 100-fold, from about 20-fold to about 500-fold, from about 20-fold to about 1,000-fold, from about 20-fold to about 10,000-fold, from about 50-fold to about 100-fold, from about 50-fold to about 500-fold, from about 50-fold to about 1,000-fold, from about 50-fold to about 10,000-fold, from about 100-fold to about 500-fold, from about 100-fold to about 1,000-fold, from about 100-fold to about 10,000-fold, from about 500-fold to about 1,000-fold, from about 500-fold to about 10,000-fold, or from about 1,000-fold to about 10,000-fold.In some embodiments, the system increases measurable Giα subunit activity in the cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold. In some embodiments, the system increases measurable Giα subunit activity in the cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system increases measurable Giα subunit activity in a cell by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0053] In some embodiments, the GPCR activity comprises measurable cAMP activity. In some cases, the measurable cAMP activity comprises an increase or decrease in cAMP concentration. In some embodiments, the system increases the measurable cAMP activity in cells comprising the system compared to the cAMP activity in cells not comprising the system. In some embodiments, the system increases the measurable cAMP in cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system increases cAMP in the cells by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5-fold, about 0.5-fold to about 10-fold, times, about 0.5 times to about 20 times, about 0.5 times to about 50 times, about 0.5 times to about 100 times, about 0.5 times to about 500 times, about 0.5 times to about 1,000 times, about 0.5 times to about 10,000 times, about 1 times to about 2 times, about 1 times to about 5 times, about 1 times to about 10 times, about 1 times to about 20 times, about 1 times to about 50 times, about 1 times to about 100 times, about 1 times to about 500 times, about 1 times to about 1,000 times, about 1 times to about 10,000 times, about 2 times to about 5 times, about 2 times to about 10 times, about 2 times to about 20 times, about 2 times to about 50 times, about 2 times to about 100 times, about 2 times to about 500 times, about 2 times to about 1,000 times, about 2 times to about 10,000 times, about 5 times to about 10 times, about 5 times to about 20 times, about 5 times to about 50 times, about 5 times to about 100 times, about 5 times to about 500 times, about 5 times to about 1,000 times, about 5 times to about 10,000 times, about 10 times to about 20 times, about 10 times to about 50 times, about 10 times to about 100 times, about 10 times to about 500 times, about 10 times to about 1,000 times, about 10 times to about 10,000 times, about 20 times from about 50-fold, from about 20-fold to about 100-fold, from about 20-fold to about 500-fold, from about 20-fold to about 1,000-fold, from about 20-fold to about 10,000-fold, from about 50-fold to about 100-fold, from about 50-fold to about 500-fold, from about 50-fold to about 1,000-fold, from about 50-fold to about 10,000-fold, from about 100-fold to about 500-fold, from about 100-fold to about 1,000-fold, from about 100-fold to about 10,000-fold, from about 500-fold to about 1,000-fold, from about 500-fold to about 10,000-fold, or from about 1,000-fold to about 10,000-fold. In some embodiments, the system increases cAMP in a cell by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system increases cAMP in the cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system increases cAMP in the cells by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0054] In some embodiments, the GPCR activity comprises measurable reverse cAMP activity. In some embodiments, the system increases the measurable reverse cAMP activity in cells comprising the system compared to the reverse cAMP activity in cells not comprising the system. In some embodiments, the system increases the reverse cAMP in the cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system increases reverse cAMP by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5-fold, about 0.5-fold to about 10 ... 0.5x to about 20x, about 0.5x to about 50x, about 0.5x to about 100x, about 0.5x to about 500x, about 0.5x to about 1,000x, about 0.5x to about 10,000x, about 1x to about 2x, about 1x to about 5x, about 1x to about 10x, about 1x to about 20x, about 1x to about 50x, about 1x to about 100x, about 1x to about 500x, about 1x to about 1,000x, about 1x to about 10,000x, about 2x to about 5x, about 2x to about 10x, about 2x to about 20x, about 2x to about 5 0x, about 2x to about 100x, about 2x to about 500x, about 2x to about 1,000x, about 2x to about 10,000x, about 5x to about 10x, about 5x to about 20x, about 5x to about 50x, about 5x to about 100x, about 5x to about 500x, about 5x to about 1,000x, about 5x to about 10,000x, about 10x to about 20x, about 10x to about 50x, about 10x to about 100x, about 10x to about 500x, about 10x to about 1,000x, about 10x to about 10,000x, about 20x to about 50-fold, about 20-fold to about 100-fold, about 20-fold to about 500-fold, about 20-fold to about 1,000-fold, about 20-fold to about 10,000-fold, about 50-fold to about 100-fold, about 50-fold to about 500-fold, about 50-fold to about 1,000-fold, about 50-fold to about 10,000-fold, about 100-fold to about 500-fold, about 100-fold to about 1,000-fold, about 100-fold to about 10,000-fold, about 500-fold to about 1,000-fold, about 500-fold to about 10,000-fold, or about 1,000-fold to about 10,000-fold. In some embodiments, the system increases reverse cAMP by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system increases reverse cAMP by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system increases reverse cAMP by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0055] In some embodiments, the system increases the measurable ratio between GPCR activity and background during measurement of GPCR activity in cells that include the system, as compared to the measurable ratio between GPCR activity and background in cells that do not include the system, in some embodiments, the system increases the measurable ratio between GPCR activity and background in the cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system increases a measurable ratio between GPCR activity in a cell and background by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 10 ... to about 5 times, about 0.5 times to about 10 times, about 0.5 times to about 20 times, about 0.5 times to about 50 times, about 0.5 times to about 100 times, about 0.5 times to about 500 times, about 0.5 times to about 1,000 times, about 0.5 times to about 10,000 times, about 1 times to about 2 times, about 1 times to about 5 times, about 1 times to about 10 times, about 1 times to about 20 times, about 1 times to about 50 times, about 1 times to about 100 times, about 1 times to about 500 times, about 1 times to about 1,000 times, about 1 times to about 10,000 times, about 2 times to about 5 times, about 2 times to about 1 ... to about 20 times, about 2 times to about 50 times, about 2 times to about 100 times, about 2 times to about 500 times, about 2 times to about 1,000 times, about 2 times to about 10,000 times, about 5 times to about 10 times, about 5 times to about 20 times, about 5 times to about 50 times, about 5 times to about 100 times, about 5 times to about 500 times, about 5 times to about 1,000 times, about 5 times to about 10,000 times, about 10 times to about 20 times, about 10 times to about 50 times, about 10 times to about 100 times, about 10 times to about 500 times, about 10 times to about 1,000 times, about 10 times to about 10,000 times , about 20-fold to about 50-fold, about 20-fold to about 100-fold, about 20-fold to about 500-fold, about 20-fold to about 1,000-fold, about 20-fold to about 10,000-fold, about 50-fold to about 100-fold, about 50-fold to about 500-fold, about 50-fold to about 1,000-fold, about 50-fold to about 10,000-fold, about 100-fold to about 500-fold, about 100-fold to about 1,000-fold, about 100-fold to about 10,000-fold, about 500-fold to about 1,000-fold, about 500-fold to about 10,000-fold, or about 1,000-fold to about 10,000-fold. In some embodiments, the system increases the ratio of GPCR activity in a cell to background by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system increases the ratio of GPCR activity in the cell to background by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system increases the ratio of GPCR activity in the cell to background by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0056] In some embodiments, the system reduces background during measurement of GPCR activity in cells comprising the system compared to background in cells not comprising the system, hi some embodiments, background in cells comprising the system is reduced by at least about 0.1-fold to about 10,000-fold during measurement of GPCR activity.In some embodiments, the background in the cells comprising the system is at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5x, about 0.5x to about 10x, about 0.5x to about 20x, about 0.5x to about 50x, about 0.5x to about 100x, about 0.5x to about 500x, about 0.5x to about 1,000x, about 0.5x to about 10,000x, about 1x to about 2x, about 1x to about 5x, about 1x to about 10x, about 1x to about 20x, about 1x to about 50x, about 1x to about 100x, about 1x to about 500x, about 1x to about 1,000x, about 1x to about 10,000x, about 2x to about 5x, about 2x to about 10x, about 2x to About 20x, about 2x to about 50x, about 2x to about 100x, about 2x to about 500x, about 2x to about 1,000x, about 2x to about 10,000x, about 5x to about 10x, about 5x to about 20x, about 5x to about 50x, about 5x to about 100x, about 5x to about 500x, about 5x to about 1,000x, about 5x to about 10,000x, about 10x to about 20x, about 10x to about 50x, about 10x to about 100x, about 10x to about 500x, about 10x to about 1,000x, about 10x to about 10,000x , about 20-fold to about 50-fold, about 20-fold to about 100-fold, about 20-fold to about 500-fold, about 20-fold to about 1,000-fold, about 20-fold to about 10,000-fold, about 50-fold to about 100-fold, about 50-fold to about 500-fold, about 50-fold to about 1,000-fold, about 50-fold to about 10,000-fold, about 100-fold to about 500-fold, about 100-fold to about 1,000-fold, about 100-fold to about 10,000-fold, about 500-fold to about 1,000-fold, about 500-fold to about 10,000-fold, or about 1,000-fold to about 10,000-fold. In some embodiments, the background in cells comprising the system is reduced by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold during measurement of GPCR activity.In some embodiments, the background in cells comprising the system is reduced by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold during measurement of GPCR activity. In some embodiments, the background in cells comprising the system is reduced by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold during measurement of GPCR activity.

[0057] In some embodiments, the system reduces the measurable coefficient of variation of measurements of GPCR activity in cells that include the system compared to the measurable coefficient of variation of measurements of GPCR activity in cells that do not include the system, hi some embodiments, the system reduces the measurable coefficient of variation of measurements of GPCR activity in cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system increases the measurable coefficient of variation of a measure of GPCR activity in a cell by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5 ... times, about 0.5 times to about 10 times, about 0.5 times to about 20 times, about 0.5 times to about 50 times, about 0.5 times to about 100 times, about 0.5 times to about 500 times, about 0.5 times to about 1,000 times, about 0.5 times to about 10,000 times, about 1 times to about 2 times, about 1 times to about 5 times, about 1 times to about 10 times, about 1 times to about 20 times, about 1 times to about 50 times, about 1 times to about 100 times, about 1 times to about 500 times, about 1 times to about 1,000 times, about 1 times to about 10,000 times, about 2 times to about 5 times, about 2 times to about 10 times, about 2 times to about 20x, about 2x to about 50x, about 2x to about 100x, about 2x to about 500x, about 2x to about 1,000x, about 2x to about 10,000x, about 5x to about 10x, about 5x to about 20x, about 5x to about 50x, about 5x to about 100x, about 5x to about 500x, about 5x to about 1,000x, about 5x to about 10,000x, about 10x to about 20x, about 10x to about 50x, about 10x to about 100x, about 10x to about 500x, about 10x to about 1,000x, about 10x to about 10,000x, about 20-fold to about 50-fold, about 20-fold to about 100-fold, about 20-fold to about 500-fold, about 20-fold to about 1,000-fold, about 20-fold to about 10,000-fold, about 50-fold to about 100-fold, about 50-fold to about 500-fold, about 50-fold to about 1,000-fold, about 50-fold to about 10,000-fold, about 100-fold to about 500-fold, about 100-fold to about 1,000-fold, about 100-fold to about 10,000-fold, about 500-fold to about 1,000-fold, about 500-fold to about 10,000-fold, or about 1,000-fold to about 10,000-fold. In some embodiments, the system reduces the measurable coefficient of variation of a measurement of GPCR activity in a cell by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system reduces the measurable coefficient of variation of measurements of GPCR activity in cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system reduces the measurable coefficient of variation of measurements of GPCR activity in cells by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0058] In some embodiments, the system reduces measurable false positives in measurements of GPCR activity in cells that include the system compared to measurable false positives in measurements of GPCR activity in cells that do not include the system, hi some embodiments, the system reduces measurable false positives in measurements of GPCR activity in cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system reduces measurable false positives in a measurement of GPCR activity in a cell by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5-fold , about 0.5 to about 10 times, about 0.5 to about 20 times, about 0.5 to about 50 times, about 0.5 to about 100 times, about 0.5 to about 500 times, about 0.5 to about 1,000 times, about 0.5 to about 10,000 times, about 1 to about 2 times, about 1 to about 5 times, about 1 to about 10 times, about 1 to about 20 times, about 1 to about 50 times, about 1 to about 100 times, about 1 to about 500 times, about 1 to about 1,000 times, about 1 to about 10,000 times, about 2 to about 5 times, about 2 to about 10 times, about 2 to about 2 times 0x, about 2x to about 50x, about 2x to about 100x, about 2x to about 500x, about 2x to about 1,000x, about 2x to about 10,000x, about 5x to about 10x, about 5x to about 20x, about 5x to about 50x, about 5x to about 100x, about 5x to about 500x, about 5x to about 1,000x, about 5x to about 10,000x, about 10x to about 20x, about 10x to about 50x, about 10x to about 100x, about 10x to about 500x, about 10x to about 1,000x, about 10x to about 10,000x, about from about 20-fold to about 50-fold, from about 20-fold to about 100-fold, from about 20-fold to about 500-fold, from about 20-fold to about 1,000-fold, from about 20-fold to about 10,000-fold, from about 50-fold to about 100-fold, from about 50-fold to about 500-fold, from about 50-fold to about 1,000-fold, from about 50-fold to about 10,000-fold, from about 100-fold to about 500-fold, from about 100-fold to about 1,000-fold, from about 100-fold to about 10,000-fold, from about 500-fold to about 1,000-fold, from about 500-fold to about 10,000-fold, or from about 1,000-fold to about 10,000-fold. In some embodiments, the system reduces false positives in measurements of GPCR activity in cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system reduces false positives in measurements of GPCR activity in cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system reduces false positives in measurements of GPCR activity in cells by at least up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0059] In some embodiments, the system reduces the measurable false negatives of measurements of GPCR activity in cells that include the system compared to the measurable false negatives of measurements of GPCR activity in cells that do not include the system, hi some embodiments, the system reduces the measurable false negatives of measurements of GPCR activity in cells by at least about 0.1-fold to about 10,000-fold.In some embodiments, the system reduces measurable false negatives in a measurement of GPCR activity in a cell by at least about 0.1-fold to about 0.5-fold, about 0.1-fold to about 1-fold, about 0.1-fold to about 2-fold, about 0.1-fold to about 5-fold, about 0.1-fold to about 10-fold, about 0.1-fold to about 20-fold, about 0.1-fold to about 50-fold, about 0.1-fold to about 100-fold, about 0.1-fold to about 500-fold, about 0.1-fold to about 1,000-fold, about 0.1-fold to about 10,000-fold, about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 5-fold , about 0.5 to about 10 times, about 0.5 to about 20 times, about 0.5 to about 50 times, about 0.5 to about 100 times, about 0.5 to about 500 times, about 0.5 to about 1,000 times, about 0.5 to about 10,000 times, about 1 to about 2 times, about 1 to about 5 times, about 1 to about 10 times, about 1 to about 20 times, about 1 to about 50 times, about 1 to about 100 times, about 1 to about 500 times, about 1 to about 1,000 times, about 1 to about 10,000 times, about 2 to about 5 times, about 2 to about 10 times, about 2 to about 2 times 0x, about 2x to about 50x, about 2x to about 100x, about 2x to about 500x, about 2x to about 1,000x, about 2x to about 10,000x, about 5x to about 10x, about 5x to about 20x, about 5x to about 50x, about 5x to about 100x, about 5x to about 500x, about 5x to about 1,000x, about 5x to about 10,000x, about 10x to about 20x, about 10x to about 50x, about 10x to about 100x, about 10x to about 500x, about 10x to about 1,000x, about 10x to about 10,000x, about from about 20-fold to about 50-fold, from about 20-fold to about 100-fold, from about 20-fold to about 500-fold, from about 20-fold to about 1,000-fold, from about 20-fold to about 10,000-fold, from about 50-fold to about 100-fold, from about 50-fold to about 500-fold, from about 50-fold to about 1,000-fold, from about 50-fold to about 10,000-fold, from about 100-fold to about 500-fold, from about 100-fold to about 1,000-fold, from about 100-fold to about 10,000-fold, from about 500-fold to about 1,000-fold, from about 500-fold to about 10,000-fold, or from about 1,000-fold to about 10,000-fold. In some embodiments, the system reduces false negatives in measurements of GPCR activity in cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.In some embodiments, the system reduces false negatives in measurements of GPCR activity in cells by at least about 0.1-fold, about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1,000-fold. In some embodiments, the system reduces false negatives in measurements of GPCR activity in cells by at least and up to about 0.5-fold, about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold, or about 10,000-fold.

[0060] Response element regulated promoter Response elements are short sequences of DNA in gene promoter regions that can bind to certain transcription factors to regulate the transcription of genes. Certain response elements are specific to certain promoters. Some response elements can be bound by endogenous transcription factors. Multiple copies of the same response element can be located in different parts of nucleotide sequences and activate different genes in response to the same stimulus. Non-limiting examples of response elements that can be incorporated into the system described herein include cAMP response element (CRE), B recognition element, AhR response element, dioxin response element or xenobiotic response element, HIF response element, hormone response element, serum response element, retinoic acid response element, peroxisome proliferator hormone response element, metal response element, DNA damage response element, IFN-stimulated response element, ROR response element, glucocorticoid response element, calcium response element CaRE1, antioxidant response element, p53 response element, thyroid hormone response element, growth hormone response element, sterol response element, polycomb response element, and vitamin D response element.

[0061] A response element-regulated promoter nucleotide sequence is a region of nucleic acid that contains one or more response elements that help recruit promoters and other molecules to regulate gene transcription. Cells contain many response element-regulated nucleotide sequences that utilize endogenous proteins to regulate gene transcription. In situations where endogenous response element-regulated promoter nucleotide sequences directly regulate reporter transcription, there is a high level of background signal due to the presence of endogenous promoters. A system that regulates reporter transcription by a transcription factor that is not endogenous to the cell that contains the system has advantages over a system that regulates reporter transcription by endogenous transcription factors. One advantage of such a system is that the reporter generates low background.

[0062] Reporter The reporter nucleic acid minimally comprises a regulatory element that can be bound by a transcription factor associated with GPCR activity (e.g., a cAMP response element (CRE) binding protein) and a nucleotide sequence encoding the reporter. The nucleotide sequence encoding the reporter is downstream of the regulatory element that can be bound by a transcription factor associated with GPCR activity. The transcription factor associated with GPCR activity regulates the expression of the reporter.

[0063] In certain embodiments, the nucleotide sequence encoding the reporter comprises a reporter gene. In certain embodiments, the reporter gene encodes a reporter selected from a fluorescent protein, a luciferase protein, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, and secreted placental alkaline phosphatase. These reporter proteins can be assayed for specific enzymatic activity or, in the case of fluorescent reporters, for fluorescence emission. In certain embodiments, the fluorescent protein comprises a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), or a cyan fluorescent protein (CFP).

[0064] In certain embodiments, the nucleotide sequence encoding the reporter gene comprises a nucleotide sequence encoding a unique sequence identifier (UMI). In certain embodiments, the UMI is unique to the test polypeptide, and the test polypeptide is encoded by the reporter nucleic acid. Generally, the UMI is between 8 and 20 nucleotides in length, but may be longer. In certain embodiments, the UMI is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides in length. In certain embodiments, the UMI is 8 nucleotides in length. In certain embodiments, the UMI is 9 nucleotides in length. In certain embodiments, the UMI is 10 nucleotides in length. In certain embodiments, the UMI is 11 nucleotides in length. In certain embodiments, the UMI is 12 nucleotides in length. In certain embodiments, the UMI is 13 nucleotides in length. In certain embodiments, the UMI is 14 nucleotides in length. In certain embodiments, the UMI is 15 nucleotides in length. In certain embodiments, the UMI is 16 nucleotides in length. In certain embodiments, the UMI is 17 nucleotides in length. In certain embodiments, the UMI is 18 nucleotides in length. In certain embodiments, the UMI is 19 nucleotides in length. In certain embodiments, the UMI is 20 nucleotides in length. In certain embodiments, the UMI is more than 20 nucleotides in length.

[0065] The system described herein can utilize a number of different regulatory sequences that control reporter gene activation by binding with transcription factors associated with GPCR activity (e.g., CREB). The regulatory sequence is one that can be bound by the transcription factor associated with GPCR activity. Generally, the regulatory sequence is configured to be 5' of the UMI, the reporter gene, or both. In certain embodiments, the regulatory sequence includes a Gal4-, PPR1-, or LexA-UAS that can be bound by the transcription factor associated with GPCR activity.

[0066] In certain embodiments, the reporter comprises a fluorescent protein, a luciferase protein, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, or secreted placental alkaline phosphatase, and a UMI. In certain embodiments, the UMI is encoded 5' of the reporter nucleic acid of the fluorescent protein, the luciferase protein, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, or secreted placental alkaline phosphatase. In certain embodiments, the nucleotide sequence encoding the fluorescent protein, the luciferase protein, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, or secreted placental alkaline phosphatase is 5' of the UMI.

[0067] The transcription of the UMI indicates the association of the reporter with a particular system, so the UMI allows for multiplexing of different systems within the same assay. The UMI may be of any length that allows for sufficient diversity to allow multiplexing of different systems within the same assay. The length must be sufficient to differentiate at least 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 targets. In certain embodiments, the different systems are present in different cells. In certain embodiments, the different systems are present in the same cell. The reporter element may further include a 5'UTR, a 3'UTR, or both. The UTR may be heterologous to the reporter element.

[0068] Reporter activation Activation of reporter molecules can be determined using standard assays to detect luciferase protein, β-galactosidase protein, β-glucuronidase protein, chloramphenicol acetyltransferase protein, secreted placental alkaline phosphatase protein. In general, there are enzyme assays that generate detectable signals based on protein enzyme activity against a substrate. For example, luciferase expression can be measured by a luminometer when luciferase substrate is present. Fluorescent reporters do not require substrates, and signals can be measured by fluorescent microscopy or fluorescent plate readers. Fluorescent reporters are particularly useful for measuring reporter activation in living cells.

[0069] In some embodiments, the reporter molecule comprises a unique RNA sequence, such as a unique molecular identifier (UMI), also referred to as an index sequence or barcode sequence. Reporter activation can be measured by any suitable method that allows for sequencing of the unique RNA sequence, preferably by a method that allows for sequencing in a multiplexed manner. Such methods include high throughput sequencing methods that can generate information on at least about 100,000, 1,000,000, 10,000,000, or 100,000,000 DNA or RNA bases in a 24 hour period. In certain embodiments, next generation sequencing technology is used to sequence the unique RNA sequence. Next generation sequencing encompasses many types of sequencing, such as pyrosequencing, sequencing by synthesis, single molecule sequencing, second generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization. Next generation sequencing platforms include those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression, or "DGE").Next generation sequencing methods include, but are not limited to, (1) the methods and instruments described in Margulies et al., Nature (2005) 437:376-380 (2005), and U.S. Pat. Nos. 7,244,559, 7,335,762, 7,211,390, 7,244,567, 7,264,929, and 7,323,305. (2) Helicos Biosciences Corporation (Cambridge, Massachusetts), as described in U.S. Provisional Patent Application No. 11 / 167046, and U.S. Patent Nos. 7,501,245, 7,491,498, 7,276,720, and U.S. Patent Application Publication Nos. 20090061439, 20080087826, 20060286566, 20060024711, 20060024678, 20080213770, and 20080103058; (3) Applied Biosystems (e.g., SOLiD Sequencing); (4) Dover Systems (e.g., Polonator G.007 sequencing), (5) Illumina, Inc., as described in U.S. Pat. Nos. 5,750,341, 6,306,597, and 5,969,119, and (6) Pacific Biosciences, Inc., as described in U.S. Pat. Nos. 7,462,452, 7,476,504, 7,405,281, 7,170,050, 7,462,468, 7,476,503, 7,315,019, 7,302,146, 7,313,308, and U.S. Patent Application Publication Nos. 20090029385, 20090068655, 20090024331, and 20080206764. Such methods and devices include, but are not limited to, those commercially available from Biosciences, Inc. Such methods and devices are presented herein by way of example and are not intended to be limiting.

[0070] cell Cells useful for the systems and methods described herein are generally those that can be easily transfected with one or more of the nucleic acids described herein. The nucleic acids of the systems that code for regulatory, effector, and / or reporter elements can be transfected or transduced into suitable cell lines using methods known in the art, such as calcium phosphate transfection, lipo-based transfection (e.g., Lipofectamine™, Lipofectamine-2000™, Lipofectamine-3000™, or Fugene® HD), electroporation, or viral transduction. The cells can also be a population of cells of the same type grown to confluence or near confluence in a suitable tissue culture vessel.

[0071] In certain embodiments, the cells used herein comprise stable integration of any of the nucleic acids encoding regulatory elements, encoding effectors, nucleic acids comprising reporter elements, or combinations thereof. Stable cell lines can be generated from the cells described herein using random integration of linear plasmids, viral integration, or transposon-directed integration, or directed integration, for example, using site-specific recombination between AttP and AttB sites. In certain embodiments, any of the nucleic acids are integrated into a safe landing site, such as the AAVS1 site.

[0072] In some embodiments, the cells described herein comprise a nucleic acid stably integrated into the genome of the cell. In some embodiments, the cells described herein comprise a nucleic acid stably integrated into the genome of the cell encoding a regulatory element. In some embodiments, the cells described herein comprise a nucleic acid stably integrated into the genome of the cell encoding at least one effector described herein. In some embodiments, the effector is a GPCR effector. In some embodiments, the GPCR effector is an adenylyl cyclase selected from the group of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10. In some embodiments, the cells comprise a stably integrated nucleic acid encoding a regulatory element for modulating expression of the effector. In some embodiments, the cells comprise a stably integrated nucleic acid encoding a regulatory element for upregulating an effector described herein. In some embodiments, the cells comprise a stably integrated nucleic acid encoding a regulatory element for upregulating ADCY6. In some embodiments, the cells comprise a stably integrated nucleic acid encoding ADCY6. In some cases, the cells contain a stably integrated nucleic acid encoding a regulatory element for downregulating an effector described herein. In some cases, the cells contain a stably integrated nucleic acid encoding a regulatory element for downregulating ADCY3.

[0073] In certain embodiments, the cell or cell population used in the system is a eukaryotic cell. In certain embodiments, the cell or cell population is a mammalian cell. In certain embodiments, the cell or cell population is a human cell. In certain embodiments, the cell or cell population is selected from the group consisting of SH-SY5Y (human neuroblastoma), Hep G2 (human white human hepatocellular carcinoma), 293 (also known as HEK 293) (human fetal kidney), RAW 264.7 (mouse monocyte macrophage), HeLa (human cervical epithelioid carcinoma), MRC-5 (PD 19) (human fetal lung), A2780 (human ovarian carcinoma), CACO-2 (human white human colon adenocarcinoma), THP 1 (human monocytic leukemia), A549 (human white human lung carcinoma), MRC-5 (PD 30) (human fetal lung), MCF7 (human white human mammary adenocarcinoma), SNL 76 / 7 (mouse cell line SIM embryonic fibroblast), C2C12 (mouse C3H myoblast), Jurkat E6.1 (human leukemic T-cell lymphoblasts), U937 (human white histiocytic lymphoma), L929 (mouse C3H / An connective tissue), 3T3 L1 (mouse embryo), HL60 (human white myeloid leukemia), PC-12 (rat adrenal pheochromocytoma), HT29 (human white colon adenocarcinoma), OE33 (human white esophageal carcinoma), OE19 (human white esophageal carcinoma), NIH 3T3 (mouse Swiss NIH embryo), MDA-MB-231 (human white breast adenocarcinoma), K562 (human white chronic myeloid leukemia), U-87 MG (human glioblastoma astrocytoma), MRC-5 (PD 25) (human fetal lung), A2780cis (human ovarian carcinoma), B9 (mouse B cell hybridoma), CHO-K1, U2OS (Chinese hamster ovary), MDCK (cocker spaniel canine kidney), 1321N1 (human brain astrocytoma), A431 (human squamous cell carcinoma), ATDC5 (derived from mouse 129 teratocarcinoma AT805), RCC4 PLUS VECTOR ALONE (renal cell carcinoma cell line RCC4 stably transfected with an empty expression vector, pcDNA3, conferring neomycin resistance), HUVEC (S200-05n) (prescreened human umbilical vein endothelial cells (HUVEC)), neonate, Vero (African green monkey kidney), RCC4 PLUS VHL (renal cell carcinoma cell line RCC4 stably transfected with pcDNA3-VHL), Fao (rat hepatocellular carcinoma), J774A.1 (mouse BALB / c monocyte-macrophage), MC3T3-E1 (mouse C57BL / 6 calvaria), and J774.2 (mouse BALB / c monocyte-macrophage), PNT1A (SV40-immortalized postpubertal human normal prostate), U-2 OS (human osteosarcoma), HCT 116 (human colon cancer), MA104 (African green monkey kidney), BEAS-2B (human normal bronchial epithelium), NB2-11 (rat lymphoma), BHK 21 (clone 13) (Syrian hamster kidney), NS0 (mouse myeloma), Neuro 2a (albino mouse neuroblastoma), SP2 / 0-Ag14, mouse x mouse non-producing myeloma, T47D (human breast tumor), 1301 (human T-cell leukemia), MDCK-II (cocker spaniel canine kidney), PNT2 (SV40 immortalized human normal prostate), PC-3 (human Caucasian prostate cancer), TF1 (human erythroleukemia), COS-7 (SV40 transformed African green monkey kidney), MDCK (cocker spaniel canine kidney), HUVEC (200-05n), human umbilical vein endothelial cells (HUVEC), neonate, NCI-H322 (human Caucasian bronchioloalveolar carcinoma), SK.N.SH (human Caucasian neuroblastoma), LNCaP.FGC (human Caucasian prostate cancer), OE21 (human Caucasian esophageal squamous cell carcinoma), PSN1 (human pancreatic adenocarcinoma), ISHIKAWA (human Asian endometrial adenocarcinoma), MFE-280 (human Caucasian endometrial adenocarcinoma), MG-63 (human osteosarcoma), RK 13 (BVDV-negative rabbit kidney), EoL-1 cells (human eosinophilic leukemia), VCaP (human prostate cancer metastasis), tsA201 (human embryonic kidney transformed with SV40), CHO (Chinese hamster ovary), HT 1080 (human fibrosarcoma), PANC-1 (human albino pancreas), Saos-2 (human primary osteogenic sarcoma), fibroblast growth medium (116K-500), fibroblast growth medium kit, ND7 / 23 (mouse neuroblastoma x rat neuron hybrid), SK-OV-3 (human albino ovarian adenocarcinoma), COV434 (human ovarian granulosa tumor), Hep 3B (human hepatocellular carcinoma), Vero (WHO) (African green monkey kidney), Nthy-ori 3-1 (human thyroid follicular epithelium, U373 MG (Uppsala) (human glioblastoma astrocytoma), A375 (human malignant melanoma), AGS (human albino gastric adenocarcinoma), CAKI 2 (human albino renal cell carcinoma), COLO 205 (human albino colon adenocarcinoma), COR-L23 (human albino large cell lung carcinoma), IMR 32 (human albino neuroblastoma), QT 35 (Japanese quail fibrosarcoma), WI 38 (human albino fetal lung), HMVII (human vaginal malignant melanoma), HT55 (human colon carcinoma), TK6 (human lymphoblastoid, thymidine kinase heterozygous), SP2 / 0-AG14 (ACFREE) (non-secreting, serum-free, animal component (AC)-free mouse x mouse hybridoma), AR42J, or rat pancreatic exocrine tumor, or any combination thereof.

[0074] In certain embodiments, the cell or cell line comprises high steady-state reporter activity. In certain embodiments, high steady-state reporter activity is at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% greater than the background level of reporter activity observed in a cell or cell line that does not contain a reporter. In certain embodiments, high steady-state reporter activity is at least about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% greater than the background level of reporter activity observed in a cell or cell line that does not contain the system described herein. For such comparisons, generally the cell or cell line used as a comparator is the parent of the cell line that comprises the line (eg, HEK293 that comprises the line versus HEK293 that does not).

[0075] In certain embodiments, the cell or cell line comprises high steady-state reporter activity. In certain embodiments, high steady-state reporter activity is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 32-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold, 750-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or 20,000-fold greater than background reporter activity, which is the level of reporter activity observed in a cell or cell line that does not contain a reporter. In certain embodiments, the high steady-state reporter activity is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 32-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold, 750-fold, 1,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, or 20,000-fold greater than background, which is the level of reporter activity observed in a cell or cell line that does not contain the system described herein. In certain embodiments, the high steady-state reporter activity is at least about 30-fold greater than background, which is the level of reporter activity observed in a cell or cell line that does not contain the system described herein. For such comparisons, generally the cell or cell line used as a comparator is the parent of the cell line that comprises the line (eg, HEK293 that comprises the line versus HEK293 that does not).

[0076] In certain embodiments, the cell or cell line comprises a low-variable stationary reporter activity. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.6. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.5. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.4. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.3. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.2. In certain embodiments, the low-variable stationary reporter activity has a biological coefficient of variation of less than about 0.1.

[0077] Methods for Measuring GPCR Activity The system described herein can be effectively used with various methods for measuring GPCR activity.The system is useful in methods for measuring GPCR activity of cell signaling pathways both at steady state and in response to physical or chemical stimuli.When reporter element contains UMI sequence that binds to specific reporter element, the system can be deployed in multiplex assay.

[0078] In one non-limiting and illustrative example, a plurality of cells are incubated in one well of a multi-well plate. The plurality of cells are transfected with at least one of the nucleic acids described herein. In some embodiments, the nucleic acid encodes a regulatory element described herein. In some embodiments, the nucleic acid encodes an effector described herein. In some embodiments, the nucleic acid encodes a reporter described herein. The transfected cells are then contacted with a ligand for the GPCR. After a sufficient time for the reporter to be expressed, cell lysates are harvested and activation of the reporter gene is measured. In this example, an increase in the presence of the reporter gene indicates that the ligand has bound or complexed with the GPCR, causing an increase in GPCR activity. In some cases, an increase in the presence of the reporter gene indicates that the ligand has bound or complexed with the GPCR, causing an increase in GPCR activity. i In some embodiments, the transfected cells contacted with the ligand exhibit increased GPCR activity, including α subunit activity. In some embodiments, the increased presence of the reporter gene indicates that the ligand has bound or complexed with the GPCR, causing an increase in GPCR activity, including cAMP activity. In some embodiments, the increased presence of the reporter gene indicates that the ligand has bound or complexed with the GPCR, causing an increase in GPCR activity, including reverse cAMP activity. In some embodiments, the transfected cells contacted with the ligand exhibit increased GPCR activity, compared to GPCR activity of a cell not transfected with a system described herein. In some embodiments, the transfected cells contacted with the ligand exhibit increased GPCR activity, compared to GPCR activity of a cell not transfected with a system described herein. iIn some embodiments, the transfected cells contacted with the ligand exhibit increased cAMP activity compared to the GPCR activity of a cell not transfected with the system described herein. In some embodiments, the transfected cells contacted with the ligand exhibit increased reverse cAMP activity compared to the GPCR activity of a cell not transfected with the system described herein.

[0079] In some embodiments, the reporter gene comprises an enzyme that generates a detectable signal upon interaction with a substrate, and standard assays known in the art can be used to quantify the activation of the reporter gene. In some embodiments, the reporter gene comprises a fluorescent molecule, and the activation of the reporter gene can be measured by a fluorescent microscope or a fluorescent plate reader, and may not require cell lysis. The fluorescent molecule is useful for measuring the activation of the reporter in live cells. In some embodiments, the reporter gene comprises a UMI, the mRNA is reverse transcribed, and the sequencing of the UMI is performed by next-generation sequencing technology.

[0080] In certain embodiments, the assay is performed in a multi-well format, such as a 6-, 12-, 24-, 48-, 96-, or 384-well format. In certain embodiments, each well is provided with a different test chemical (e.g., a ligand), or duplicate, triplicate, or quadruplicate wells are provided with test chemicals. The assay may also include one or more positive control wells, or one negative control well. In some embodiments, the method includes measuring GPCR activity in a cell population transfected with at least one nucleic acid described herein. The cell population transfected with at least one nucleic acid of the present disclosure may be of any size. In certain embodiments, the cell population includes 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 or more cells. In certain embodiments, at least about 1,000 or more cells are transfected with at least one nucleic acid of the system described herein. In certain embodiments, at least about 10,000 or more cells are transfected with at least one nucleic acid of the system described herein. In certain embodiments, at least about 100,000 or more cells are transfected with at least one nucleic acid of the system described herein. In certain embodiments, at least about 1,000,000 or more cells are transfected with at least one nucleic acid of the system described herein. In certain embodiments, at least about 10,000,000 or more cells are transfected with at least one nucleic acid of the system described herein. In certain embodiments, the system of the present disclosure can be utilized in multi-well plate experiments. Non-limiting examples of multi-well plates compatible with the system of the present disclosure include 6, 12, 24, 48, 96, 384, or 1,536 well plates. In certain embodiments, each well of the multi-well plate contains a cell population transfected with at least one nucleic acid of the system described herein. In certain embodiments, each well of the multi-well plate contains a cell population transfected with at least one nucleic acid of the system described herein.In certain embodiments, each well contains multiple cell populations, each transfected with at least one nucleic acid of the system described herein. In certain embodiments, each well contains multiple cell populations, each transfected with at least one nucleic acid of the system described herein.

[0081] One advantage of the existing system is that libraries of cells expressing different targets can be cultured together in the same well and analyzed in parallel, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 500, 1,000 or more targets in the same experiment. This is achieved by associating targets with their unique UMIs, which can then be analyzed by high-throughput sequencing to deconvolute the data. Thus, in certain embodiments, a system is described herein that includes a plurality of mammalian cells, each of the plurality of mammalian cells includes a different GPCR target, and each of the plurality of mammalian cells includes (a) a first effector of GPCR activity, whose expression is upregulated compared to the expression of the first effector of GPCR activity in a wild-type state, and (b) a second effector of GPCR activity, whose expression is downregulated compared to the expression of the second effector of GPCR activity in a wild-type state. In certain embodiments, the plurality of mammalian cells further comprises a reporter gene comprising a unique molecular identifier (UMI) nucleic acid sequence activatable by a promoter responsive to GPCR signaling.

[0082] Thus, in certain embodiments, a system is described herein that includes a plurality of mammalian cells, each of the plurality of mammalian cells comprising a different target, each of the plurality of mammalian cells comprising (a) a first effector of target activity whose expression is upregulated compared to the expression of the first effector of target activity in a wild-type state, and (b) a second effector of target activity whose expression is downregulated compared to the expression of the second effector of target activity in a wild-type state. In certain embodiments, the plurality of mammalian cells further comprises a reporter gene comprising a nucleic acid sequence of a unique molecular identifier (UMI) that is activatable by a promoter responsive to target signaling.

[0083] In certain embodiments, a test agent, such as a ligand that complexes or binds with GPCR, is applied to cells transfected with at least one nucleic acid of the system described herein.In certain embodiments, the transcription activation level of reporter molecule is measured after contacting the cell with the test agent.In certain embodiments, the test agent is a chemical, a small molecule, a biomolecule, a polypeptide, a polynucleotide, an aptamer, or any combination thereof.In certain embodiments, a single test agent is applied to the cell population.In certain embodiments, multiple test agents are applied to the cell population.

[0084] In some embodiments, the methods described herein measure GPCR activity by contacting a ligand with a GPCR expressed on the surface of a cell comprising a system described herein. In some embodiments, the methods measure GPCR activity by contacting a plurality of ligands with a GPCR expressed on the surface of a cell comprising a system described herein. In some embodiments, the methods measure GPCR activity by contacting a ligand with a plurality of GPCRs expressed on the surface of a cell comprising a system described herein. In some embodiments, the methods measure GPCR activity by contacting a plurality of ligands with a plurality of GPCRs expressed on the surface of a cell comprising a system described herein.

[0085] In certain embodiments, the system is adapted to measure the response of GPCR to test drug or ligand.The system of the present disclosure can be adapted to use with any GPCR receptor.In certain embodiments, the system is adapted to use with GPCR receptor by utilizing cAMP response element regulated promoter. Non-limiting examples of GPCRs include 5-hydroxytryptamine receptors, acetylcholine receptors, adenosine receptors, adrenergic receptors, angiotensin receptors, apelin receptors, bile acid receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemerin receptors, chemokine receptors, cholecystokinin receptors, dopamine receptors, endothelin receptors, formyl peptide receptors, free fatty acid receptors, galanin receptors, ghrelin receptors, glycoprotein hormone receptors, gonadotropin releasing hormone receptors, GPR18, GPR55, GPR119, G protein-coupled estrogen receptors, histamine receptors, hydroxycarboxylic acid receptors, kisspeptin receptors, leukotriene receptors, LPA receptors, S1P receptors, melanin-concentrating hormone receptors, melanocortin receptors, melatonin receptors, motilin receptors, neuromedicinal receptors, and the like. Neuropeptide U receptors, Neuropeptide FF / Neuropeptide AF receptors, Neuropeptide S receptors, Neuropeptide W / Neuropeptide B receptors, Neuropeptide Y receptors, Neurotensin receptors, Opioid receptors, Opsin receptors, Orexin receptors, Oxoglutarate receptors, P2Y receptors, Platelet activating factor receptors, Prokineticin receptors, Prolactin releasing peptide receptors, Prostanoid receptors, Protease activated receptors, QRFP receptors, Relaxin family peptide receptors, Somatostatin receptors, Succinate receptors, Tachykinin receptors, Thyrotropin releasing hormone receptors, Trace amine receptors, Urotensin receptors, Vasopressin and oxytocin receptors, Calcitonin receptors, Corticotropin releasing factor receptors, Glucagon receptor family, Parathyroid hormone receptors, VIP and PACAP receptors, Calcium sensing receptors, GABA receptors B receptors, metabotropic glutamate receptors, taste 1 receptors, frizzled class receptors, adhesion class GPCRs, orphan receptors, or any combination thereof.

[0086] The nucleic acid of the present disclosure is compatible with many vectors that are common in the art.Non-limiting examples of vectors include genome-integrating vectors, episomal vectors, plasmids, viral vectors, cosmids, bacterial artificial chromosomes, and yeast artificial chromosomes.Non-limiting examples of viral vectors that are compatible with the nucleic acid of the present disclosure include vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses.In certain embodiments, the nucleic acid of the present disclosure is present in a vector that comprises a sequence that directs site-specific integration (e.g., AttP-AttB recombination) at a set of defined positions or restricted sites in the genome.

[0087] In certain embodiments, the systems described herein are incorporated into a single vector. In certain embodiments, the single vector is transiently transfected into the cells. In certain embodiments, the single vector is stably transfected into the cells.

[0088] In certain embodiments, the system is divided into two vectors. In certain embodiments, the first vector comprises a first regulatory element and a first effector, and the second vector comprises a second regulatory element for regulating the expression of the second effector. In certain embodiments, the first vector and the second vector are transiently transfected into cells. In certain embodiments, the first vector and the second vector are stably transfected into cells. In certain embodiments, the first vector is stably transfected into cells, and the second vector is transiently transfected into cells. In certain embodiments, the first vector is transiently transfected into cells, and the second vector is stably transfected into cells. In some embodiments, a separate vector containing a reporter can be transfected into cells. In some embodiments, the cells transfected with the first vector or the second vector already contain a reporter.

[0089] Vectors comprising the systems or parts thereof described herein or portions thereof can be constructed using a number of well-known molecular biology techniques. Detailed protocols for many such protocols, including amplification, cloning, mutagenesis, transformation, etc., can be found, for example, in Ausubel et al. Current Protocols in Molecular Biology (supplemented through 2012) John Wiley & Sons, New York 10 ("Ausubel"); Sambrook et al. Molecular Cloning - A Laboratory Manual (4th Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2012 ("Sambrook"); and Abelson et al. Guide to Molecular Cloning Techniques (Methods in Enzymology) volume 152 Academic Press, Inc., San Diego, CA ("Abelson").

[0090] kit Disclosed herein, in some embodiments, is a kit comprising a system described herein. Optionally, the kit comprises at least one nucleic acid of the system described herein. In some embodiments, the kit does not comprise a cell. In some embodiments, the kit comprises a cell transfected with at least a nucleic acid described herein. In some embodiments, the kit comprises a cell to be transfected with at least a nucleic acid described herein. In some embodiments, the kit disclosed herein can be used to measure GPCR activity in a cell. In some embodiments, the kit disclosed herein can be used to modulate GPCR activity in a cell. In some embodiments, the kit disclosed herein can be used to modulate G protein activity in a cell. In some embodiments, the kit disclosed herein can be used to measure GPCR activity in a cell. iIn some embodiments, the kits disclosed herein can be used to modulate α subunit activity. In some embodiments, the kits disclosed herein can be used to modulate cAMP activity in a cell. In some embodiments, the kits disclosed herein can be used to modulate reverse cAMP activity in a cell. In some embodiments, the kits disclosed herein can be used to increase GPCR activity in a cell. In some embodiments, the kits disclosed herein can be used to increase G protein activity in a cell. In some embodiments, the kits disclosed herein can be used to modulate GPCR activity in a cell. i It can be used to increase α subunit activity. In some embodiments, the kits disclosed herein can be used to increase cAMP activity in a cell. In some embodiments, the kits disclosed herein can be used to increase reverse cAMP activity in a cell.

[0091] In some embodiments, the kits disclosed herein can be used to modulate the expression of a GPCR effector in a cell. In some embodiments, the kits disclosed herein can be used to increase the expression of a GPCR effector in a cell. In some embodiments, the kits disclosed herein can be used to decrease the expression of a GPCR effector in a cell. In some embodiments, the kits disclosed herein can be used to increase the expression of a first GPCR effector in a cell or decrease the expression of a second GPCR effector in a cell. In some embodiments, the GPCR effector is adenylyl cyclase. The adenylyl cyclase can be ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, or ADCY10. In some embodiments, the kits disclosed herein can be used to increase the expression of any one of the adenylyl cyclases. In some embodiments, the kits disclosed herein can be used to increase the expression of ADCY6. In some embodiments, the kits disclosed herein can be used to reduce the expression of any one of the adenylyl cyclases. In some embodiments, the kits disclosed herein can be used to reduce the expression of ADCY3.

[0092] In some embodiments, the kit comprises the system described herein and can be used to carry out the methods described herein. The kit comprises a collection of materials or compositions including at least one of the components of the system. In other embodiments, the kit contains all the necessary and / or sufficient compositions to carry out the methods described herein, including all controls and inducers.

[0093] In some cases, the kits described herein include the systems described herein for modulating the gene activity of any one of the transgenes described herein. The exact nature of the components configured in the kit depends on its intended purpose. For example, some kits may be configured to screen for GPCR activity where a ligand binds or complexes with the GPCR. In some embodiments, the kits may be specifically configured to screen for unknown ligands. In some embodiments, the kits may be specifically configured to measure GPCR activity when stimulated by a ligand or in the absence of a ligand.

[0094] In some cases, the kit may include instructions for use. Optionally, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring devices, dressings, or other useful equipment. The materials and components combined in the kit may be stored and provided to the medical practitioner in any convenient and suitable manner that preserves their operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form and may be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging materials. As used herein, the phrase "packaging materials" refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging materials are constructed by well-known methods and preferably provide a sterile, contaminant-free environment. The packaging materials used in the kits are those that are conventionally used in gene expression assays and therapeutic administration. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of holding individual kit components.Thus, for example, packaging may be a glass vial or a pre-filled syringe that is used to contain a suitable amount of pharmaceutical composition.The packaging material has an external label that indicates the contents and / or purpose of the kit and its components.

[0095] While preferred embodiments of the system and method have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The system and method are not intended to be limited by the specific examples provided herein. Although the system and method have been described with reference to the foregoing specification, the description and illustration of the embodiments herein should not be construed in a limiting sense. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it will be understood that all aspects of the system and method are not limited to the specific depictions, configurations or relative proportions shown herein, but are determined by a variety of conditions and variables. It will be understood that various alternatives to the embodiments of the system and method described herein may be used in the practice of the disclosure. It is therefore intended that the system and method described herein shall also cover any such alternatives, modifications, variations or equivalents. The following claims define the scope of the system and method described herein, and it is intended that the system and method described herein be within the scope of these claims and that their equivalents are covered by these claims.

[0096] Embodiment Embodiment 1. A system for measuring G protein-coupled receptor (GPCR) activity, comprising a first effector of GPCR activity whose expression is upregulated compared to expression of the first effector in a wild-type state, and a second effector of GPCR activity whose expression is downregulated compared to expression of the second effector in a wild-type state.

[0097] Embodiment 2. The system of embodiment 1, wherein expression of the first effector is upregulated by contacting the GPCR with a GPCR activator.

[0098] Embodiment 3. The system of embodiment 2, wherein the GPCR activator is a GPCR agonist.

[0099] Embodiment 4. The system of embodiment 2, wherein the GPCR activator is an agonist of the first effector.

[0100] Embodiment 5. The system of embodiment 1, wherein the system comprises a first nucleic acid encoding a first effector of GPCR activity, operably linked to a regulatory element for upregulating expression of the first effector.

[0101] Embodiment 6. The system of embodiment 1, wherein expression of the second effector is downregulated by contacting the GPCR with a GPCR inhibitor.

[0102] Embodiment 7. The system of embodiment 6, wherein the GPCR inhibitor is a GPCR inverse agonist.

[0103] Embodiment 8. The system of embodiment 6, wherein the GPCR inhibitor is a GPCR antagonist.

[0104] Embodiment 9. The system of embodiment 6, wherein the GPCR inhibitor is an inverse agonist of the second effector.

[0105] Embodiment 10. The system of embodiment 6, wherein the GPCR inhibitor is an antagonist of the second effector.

[0106] Embodiment 11. The system of embodiment 1, wherein the system comprises a second nucleic acid that downregulates expression of a second effector.

[0107] Embodiment 12. The system of embodiment 1, wherein the GPCR activity comprises Giα subunit activity, Gsα subunit activity, Gqα subunit activity, or G12 / 13α subunit activity.

[0108] Embodiment 13. The system of embodiment 12, wherein the GPCR activity comprises Giα subunit activity.

[0109] Embodiment 14. The system of embodiment 13, wherein the Giα subunit activity comprises activity of Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, or Gαz.

[0110] Embodiment 15. The system of embodiment 12, wherein the GPCR activity comprises a Gsα subunit activity.

[0111] Embodiment 16. The system of embodiment 12, wherein the GPCR activity comprises Gqα subunit activity.

[0112] Embodiment 17. The system of embodiment 12, wherein said GPCR activity comprises G12 / 13 α subunit activity.

[0113] Embodiment 18. The system of embodiment 5, wherein the regulatory element is inducible.

[0114] Embodiment 19. The system of embodiment 5, wherein the regulatory element is constitutively active.

[0115] Embodiment 20. The system of any one of the previous embodiments, wherein the first effector comprises a first adenylyl cyclase.

[0116] Embodiment 21. The system of embodiment 20, wherein the first adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10.

[0117] Embodiment 22. The system of embodiment 21, wherein the first adenylyl cyclase is ADCY6.

[0118] Embodiment 23. The system of any one of the previous embodiments, wherein the second effector comprises a second adenylyl cyclase.

[0119] Embodiment 24. The system of embodiment 23, wherein the second adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10.

[0120] Embodiment 25. The system of embodiment 24, wherein the second adenylyl cyclase is ADCY3.

[0121] Embodiment 26. The system of any one of embodiments 12 to 25, wherein the second nucleic acid comprises a microRNA, an shRNA, an siRNA, a gRNA, or a combination thereof.

[0122]

[0036] Embodiment 27. The system of embodiment 26, wherein the second nucleic acid comprises a microRNA.

[0123] Embodiment 28. The system of embodiment 26, wherein the second nucleic acid comprises a gRNA.

[0124] Embodiment 29. The system of embodiment 28, further comprising a CRISPR-Cas system operably linked to the gRNA.

[0125] Embodiment 30. The system of embodiment 1, wherein the GPCR activity comprises cyclic AMP (cAMP) activity.

[0126]

[0032] Embodiment 31. The system of embodiment 1, wherein the GPCR activity comprises a change in cAMP concentration.

[0127] Embodiment 32. The system of embodiment 1, wherein the GPCR activity comprises reverse cAMP activity.

[0128] Embodiment 33. The system of embodiment 20, wherein the system increases cAMP activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to cAMP activity in cells not comprising the system.

[0129] Embodiment 34. The system of embodiment 31, wherein the system increases the cAMP concentration in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to the cAMP concentration in cells not comprising the system.

[0130] Embodiment 35. The system of embodiment 32, wherein the system increases reverse cAMP activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to reverse cAMP activity in cells not comprising the system.

[0131] Embodiment 36. The system of any one of the previous embodiments, wherein the system comprises a second regulatory element for upregulating expression of the Giα subunit.

[0132] Embodiment 37. The system of any one of the previous embodiments, wherein the system comprises at least one additional effector.

[0133] Embodiment 38. The system of embodiment 37, wherein at least one further effector is adenylyl cyclase.

[0134] Embodiment 39. The system of embodiment 37, wherein the system further comprises a second regulatory element for upregulating expression of at least one additional effector.

[0135] Embodiment 40. The system of any one of the previous embodiments, wherein the system increases the ratio between GPCR activity and background in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to the ratio between GPCR activity and background in cells not comprising the system.

[0136] Embodiment 41. The system of embodiment 40, wherein the system increases GPCR activity in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to GPCR activity in cells not comprising the system.

[0137] Embodiment 42. The system of embodiment 40, wherein the system reduces background in cells comprising the system by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to background in cells not comprising the system.

[0138] Embodiment 43. The system of any one of the previous embodiments, wherein the system comprises a reporter nucleic acid, which generates a detectable signal proportional to GPCR activity.

[0139] Embodiment 44. The system of embodiment 43, wherein the reporter nucleic acid comprises a cAMP response element (CRE) sequence.

[0140] Embodiment 45. The system of embodiment 43, wherein the reporter nucleic acid is operably linked to a unique molecular identifier (UMI) nucleic acid sequence.

[0141] Embodiment 46. The system of embodiment 43, wherein the detectable signal comprises a fluorescent signal.

[0142] Embodiment 47. The system of embodiment 43, wherein the detectable signal comprises a luminescent signal.

[0143] Embodiment 48. The system of embodiment 5, wherein the first nucleic acid comprises a gene expression cassette for upregulating expression of the first effector.

[0144] Embodiment 49. The system of embodiment 11, wherein the second nucleic acid comprises a gene expression cassette for downregulating expression of the second effector.

[0145] Embodiment 50. A cell comprising the system according to any one of embodiments 1 to 49.

[0146] Embodiment 51. The cell of embodiment 50, wherein the cell comprises a eukaryotic cell.

[0147] Embodiment 52. The cell of embodiment 50, wherein the cell comprises a mammalian cell.

[0148] Embodiment 53. The cell of embodiment 50, comprising a cell of mammalian cell origin.

[0149] Embodiment 54. The cell of embodiment 50, wherein the cell is from a cell line.

[0150] Embodiment 55. The cell of embodiment 50, wherein the cell is selected from the group consisting of a CHO-K1 cell, a COS-7 cell, and a U2OS cell.

[0151] Embodiment 56. The cell of any one of embodiments 50 to 55, wherein the system is integrated into the genome of the cell.

[0152] Embodiment 57. A cell population comprising the cells of any one of embodiments 52 to 57.

[0153] Embodiment 58. The cell population of embodiment 57, wherein the cell population comprises a population of eukaryotic cells.

[0154] Embodiment 59. The cell population of embodiment 57, wherein the cell population comprises a population of mammalian cells.

[0155] Embodiment 60. The cell population of embodiment 57, wherein the cell population comprises a population of cells derived from mammalian cells.

[0156] Embodiment 61. The cell population of embodiment 55, wherein the cell population comprises a population of cells from a cell line.

[0157] Embodiment 62. The cell population of embodiment 55, wherein the cell population comprises a population of cells selected from the group consisting of CHO-K1 cells, COS-7 cells, and U2OS cells.

[0158] Embodiment 63. A method for measuring GPCR activity, the method comprising measuring GPCR activity in a cell according to any one of embodiments 50 to 56, or in a cell population according to any one of embodiments 57 to 62.

[0159]

[0046] Embodiment 64. The method of embodiment 63, wherein the GPCR activity is a constant GPCR activity.

[0160]

[0046] Embodiment 65. The method of embodiment 63, wherein the cell of any one of embodiments 50 to 56, or the cell population of any one of embodiments 57 to 62, is not contacted with a GPCR ligand.

[0161] Embodiment 66. A method for measuring GPCR activity in a cell, the method comprising contacting a cell according to any one of embodiments 50 to 56, or a cell population according to any one of embodiments 57 to 62, with a GPCR ligand, wherein the ligand complexes with the GPCR and initiates or inhibits GPCR activity.

[0162]

[0046] Embodiment 67. The method of embodiment 66, wherein the GPCR ligand is expressed in a cell of any one of embodiments 50 to 56.

[0163] Embodiment 68. The method of embodiment 67, wherein the GPCR ligand is selected from the group consisting of a polypeptide, a non-peptide compound, and a small molecule. EXAMPLES

[0164] The following illustrative examples are representative embodiments of the stimuli, systems, and methods described herein and are not meant to be limiting in any way.

[0165] Example 1. Measurement of GPCR activity GPCR activity was measured from cells transfected with different plasmids as shown in Figure 2. The plasmid described in Figure 2A contained a gene expression cassette encoding a miRNA array that targets the endogenous adenylyl cyclase 3 (AC3) mRNA for downregulation. This cassette also contained an exogenous sequence that encoded the production of adenylyl cyclase 6 (AC6) protein. Both of these elements were under the control of the same coumarate-regulated promoter. This nucleic acid also contained a second cassette that encoded the production of a gene that confers resistance to the antibiotic blasticidin. The plasmid was stably integrated into the HEK293 cell line by co-transfection with a second plasmid containing the expression cassette and bxbi integrase. The cell line and the plasmid contained the bxbi integration sequence, which allowed the plasmid to be integrated in a single copy into a specific locus in the genome. The cell lines also contained expression cassettes encoding the production of a cumate-repressible protein, allowing cumate-inducible expression of a cumate-regulated promoter, and a reverse tetracycline transactivator (rTTA) protein, allowing doxycycline-inducible expression. Successful integration was captured by passaging the cells in medium supplemented with blasticidin.

[0166] The plasmid depicted in Figure 2B contained a gene expression cassette encoding a dCas9-BFP-KRAB fusion protein and multiple expression cassettes encoding three different RNA guides (gRNAs) targeting AC3. Downregulation was achieved by targeting dCas9-BFP-KRAB to the native AC3 genomic locus by gRNA. The plasmid also contained gene expression cassettes encoding hygromycin resistance and a sleeping beauty transposon integration sequence. Two different plasmids, each containing three guides (for a total of five unique guides) for AC3, were stably integrated into the bxbi integration cell line by co-transfecting with a plasmid encoding the expression of the sleeping beauty transposase. Successful integration was captured by passaging the cells in medium supplemented with hygromycin.

[0167] The plasmid depicted in FIG. 2C contained a gene expression cassette under a cumate-controlled promoter that codes for the production of the AC6 protein. This plasmid also contained a second gene expression cassette that codes for the production of a cumate-repressed protein that allows cumate-inducible expression of the cumate-controlled promoter, and a puromycin resistance gene. This plasmid also contained a piggybac transposon integration sequence. The plasmid was stably integrated into the HEK293 cell line described above by co-transfecting with a plasmid that codes for the expression of the piggybac transposon. Successful integration was captured by passaging the cells in medium supplemented with puromycin. The nucleic acid sequences of the plasmids in FIG. 2 can be found in SEQ ID NOs: 1-4.

[0168] Cells transfected with the second generation gene expression cassette (Figure 2A) were contacted with dopamine and forskolin (Fsk) 0.5 μM (Figure 3). As shown in Figure 3, GPCR activity associated with adrenaline, dopamine, and serotonin, which bind to the receptors ADRA2A, ADRA2C, DRD2, DRD3, DRD4, and HTR1F, respectively, was increased in cells transfected with MADSv2.5 nucleic acid. As shown in Figure 4, luciferase measurements generated from cells containing the first generation gene expression cassette (Figure 2C) show that only AC6 overexpression (AC6-OE) increased reverse CRE activity. As shown in Figure 5, platform data generated from cells containing the third generation cassette (Figure 2B, including AC3 knockdown ADCY3-KD) showed improvement compared to platform data generated from cells containing the second generation gene expression cassette, with ADCY3 knockdown leading to a decrease in measured activity.

[0169] Although the foregoing disclosure has been described in detail for clarity and understanding, it will be apparent to one skilled in the art upon reading this disclosure that various changes in form and detail can be made without departing from the disclosure. For example, all of the techniques and devices described above can be used in various combinations. All publications, patents, patent applications, and / or other documents referred to herein are incorporated by reference in their entirety for all purposes, just as if each individual publication, patent, patent application, and / or other document was individually and individually indicated to be incorporated by reference for all purposes. Definitions contained in texts incorporated by reference are excluded to the extent that they are inconsistent with definitions in this disclosure.

Claims

1. 1. A system for measuring G protein-coupled receptor (GPCR) activity in mammalian cells, comprising: a) a first effector of GPCR activity whose expression is upregulated compared to the expression of the first effector of GPCR activity in a wild-type state; b) a second effector of GPCR activity, the expression of which is downregulated compared to the expression of the second effector of GPCR activity in a wild-type state; and A system including:

2. The GPCR activity is ｉ α subunit activity, G ｓ α subunit activity, G ｑ α subunit activity, or G １２／１３ The system of claim 1 comprising an α subunit activity.

3. The system of claim 2, wherein the G i α subunit activity includes activity of Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, or Gαz.

4. The system of claim 3, wherein the GPCR activity comprises Gs α-subunit activity, Gq α-subunit activity, or G12 / 13 α-subunit activity.

5. A system described in any one of claims 1 to 4, wherein a first effector of the GPCR activity includes a first adenylyl cyclase.

6. The system described in claim 5, wherein the first adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10.

7. The system described in claim 5 or 6, wherein the first adenylyl cyclase is ADCY6.

8. A system described in any one of claims 5 to 7, wherein the first adenylyl cyclase is encoded by a nucleic acid operably linked to a promoter or enhancer that overexpresses the first adenylyl cyclase compared to a wild-type state.

9. A system described in any one of claims 1 to 8, wherein a second effector of the GPCR activity includes a second adenylyl cyclase.

10. The system described in claim 9, wherein the second adenylyl cyclase is selected from the group consisting of ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, and ADCY10.

11. The system described in claim 9 or 10, wherein the second adenylyl cyclase is ADCY3.

12. A system described in any one of claims 9 to 11, wherein the second adenylyl cyclase is targeted by one or more of microRNA, shRNA, siRNA, or a CRISPR / Cas9 complex that reduces expression of the second adenylyl cyclase compared to a wild-type state.

13. A system described in any one of claims 1 to 12, wherein the system includes a reporter nucleic acid, the reporter nucleic acid generating a detectable signal from a reporter gene that is proportional to the GPCR activity.

14. The system of claim 13, wherein the reporter nucleic acid comprises a cAMP response element (CRE) sequence operably linked to the reporter gene.

15. The system described in claim 13 or 14, wherein the reporter gene comprises a unique molecular identifier (UMI) nucleic acid sequence.

16. A system described in any one of claims 13 to 15, wherein the reporter gene comprises one or more of expression of a unique molecular identifier, a fluorescent signal, or a luminescent signal.

17. The system of any one of claims 1 to 16, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO-K1 cells, COS-7 cells, and U2OS cells.

18. A system described in any one of claims 1 to 17, wherein the system is integrated into the genome of the mammalian cell.

19. A method for measuring GPCR activity in a mammalian cell, the method comprising contacting a system described in any one of claims 1 to 18 with a GPCR ligand, wherein the ligand forms a complex with the GPCR and initiates or inhibits the GPCR activity.

20. The method of claim 19, wherein the GPCR ligand is selected from the group consisting of polypeptides and small molecules.

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