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Cloning and recombinant production of CRF receptor(s)

a technology of crf receptor and dna sequence, which is applied in the field of receptor proteins and dna sequences encoding same, can solve the problems of little known about the structure of these receptors or about the second messenger signalling system

Inactive Publication Date: 2005-08-25
SALK INST FOR BIOLOGICAL STUDIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The DNAs of the invention are useful as probes for the identification of additional members of the invention superfamily of receptor proteins, and as coding sequences which can be used for the recombinant expression of the invention receptor proteins,

Problems solved by technology

However, little is known about the structure of these receptors, or about the second messenger signalling systems that they employ.

Method used

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  • Cloning and recombinant production of CRF receptor(s)
  • Cloning and recombinant production of CRF receptor(s)
  • Cloning and recombinant production of CRF receptor(s)

Examples

Experimental program
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examples

[0127]Recombinant human (rh) activin A, rh activin B, and rh inhibin A were generously provided by Genentech, Inc. Porcine TGF-β1 was obtained from R+D Systems.

[0128] Double-stranded DNA was sequenced by the dideoxy chain termination method using the Sequenase reagents from US Biochemicals. Comparison of DNA sequences to databases was performed using the FASTA program (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)].

example i

Construction and Subdivision of AtT20 cDNA Library

[0129] Polyadenylated RNA was prepared from AtT20 cells using the Fast Track reagents from InVitrogen. cDNA was commercially synthesized and ligated into the plasmid vector pcDNAl using non-palindromic BstXI linkers, yielding a library of approximately 5×106 primary recombinants. The unamplified cDNA library was plated at 1000 clones per 100 mm plate, then scraped off the plates, frozen in glycerol and stored at −70°.

[0130] Activin suppresses adrenocorticotrophic hormone (ACTH) secretion by both primary anterior pituitary cell cultures [Vale et al., Nature 321: 776-779 (1986)] and AtT20 mouse corticotropic cells. Because AtT20 cells possess activin receptors indistinguishable from those on other cell types (based on binding affinity measurements with activin A), these cells were chosen to be the source of cDNA for transfection. A cDNA library of approximately 5×106 independent clones from AtT20 cells was constructed in the mammalia...

example ii

COS Cell Transfection

[0132] Aliquots of the frozen pools of clones from Example I were grown overnight in 3 ml cultures of terrific broth, and mini-prep DNA prepared from 1.5 ml using the alkaline lysis method [Maniatis et al. Molecular Cloning (Cold Spring Harbor Laboratory (1982)]. 1 / 10 of the DNA from a mini-prep (10 Ml of 100 Ml) was used for each transfection.

[0133] 2×10 5 COS cells were plated on chambered microscope slides (1 chamber-Nunc) that had been coated with 20 μg / ml poly-D-lysine and allowed to attach for at least 3 hours. Cells were subjected to DEAE-Dextran mediated transfection as follows. 1.5 ml of serum-free Dulbecco's Modified Eagle's medium (DME) containing 100 mM chloroquine was added to the cells. DNA was precipitated in 200 ml DME / chloroquine containing 500 mg / ml DEAE-Dextran, then added to the cells. The cells were incubated at 37° for 4 hours, then the media was removed and the cells were treated with 10% DMSO in HEPES buffered saline for 2 minutes. Fres...

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Abstract

In accordance with the present invention, there are provided novel receptor proteins characterized by having the following domains, reading from the N-terminal end of said protein: an extracellular, ligand-binding domain, a hydrophobic, trans-membrane domain, and an intracellular, receptor domain having serine kinase-like activity. The invention receptors optionally further comprise a second hydrophobic domain at the amino terminus thereof. The invention receptor proteins are further characterized by having sufficient binding affinity for at least one member of the activin / TGF-β superfamily of polypeptide growth factors such that concentrations of ≦10 nM of said polypeptide growth factor occupy ≦50% of the binding sites of said receptor protein. A presently preferred member of the invention superfamily of receptors binds specifically to activins, in preference to inhibins, transforming growth factor-β, and other non-activin-like proteins. DNA sequences encoding such receptors, assays employing same, as well as antibodies derived therefrom, are also disclosed.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 300,584, filed Sep. 2, 1994, now pending, which is a continuation of U.S. Ser. No. 07 / 880,220, filed May 8, 1992, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07 / 773,229, filed Oct. 9, 1991, now abandoned, which is, in turn, a continuation-in-part of U.S. Ser. No. 07 / 698,709, filed May 10, 1991, now abandoned.ACKNOWLEDGEMENT [0002] This invention was made with Government support under Grant Numbers HD 13527 and DK 26741, awarded by the National Institutes of Health. The Government has certain rights in this invention.FIELD OF THE INVENTION [0003] The present invention relates to receptor proteins, DNA sequences encoding same, and various uses therefor. BACKGROUND OF THE INVENTION [0004] Activins are dimeric proteins which have the ability to stimulate the production of follicle stimulating hormone (FSH) by the pituitary gland. Activins share a common subunit with inhibins, w...

Claims

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Application Information

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IPC IPC(8): A61K38/00C07K14/71
CPCA61K38/00C07K2319/00C07K14/71
Inventor MATHEWS, LAWRENCEVALE, WYLIETSUCHIDA, KUNIHIRO
Owner SALK INST FOR BIOLOGICAL STUDIES
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