GFRalpha3 and its uses

a technology of gfralpha3 and its use, which is applied in the field of identification and isolation of novel dna, can solve problems such as different malignancies and pathological disorders, and achieve the effects of facilitating identification and characterization, high throughput, and facilitating trapping and detection

Inactive Publication Date: 2005-10-06
DE SAUVAGE FREDERIC +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] In view of the surprising finding herein that the α-subunit receptor can dimerize upon ligand binding, and further that such dimerization can activate a kinase domain fused the α-subunit receptor, a method is provided herein to measure ligand-induced α-subunit receptor activation, i.e. homo-dimerizaiton or homo-oligomerization. In one embodiment is provided a sensitive, reliable assay that measures agonist- or ligand-induced α-subunit receptor activation, i.e., homo-dimerization or homo-oligomerization, preferably by measuring receptor protein tyrosine kinase (rPTK) autophosphorylation of a polypeptide fusion comprising the ligand binding domain of an α-subunit receptor and the intracellular catalytic domain of a receptor protein tyrosine kinase. The construct can further optionally comprise a flag epitope to facilitate trapping and detection of the activated (e.g., dimerized, phosphorylated) α-subunit receptor. The assay is desirably useful for qualitatively and quantitatively measuring α-subunit receptor activation as well as facilitating identification and characterization of potential agonists and antagonists for a selected α-subunit receptor. It is a further object of the invention to provide an assay which enables ligand-receptor interactions to be studied for any selected α-subunit receptor, and preferably a GFRα subunit receptor.
[0017] This assay must have a capacity for high throughput, that is

Problems solved by technology

The aberrant expression or uncontrolled regulation of any one of these receptor

Method used

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  • GFRalpha3 and its uses
  • GFRalpha3 and its uses
  • GFRalpha3 and its uses

Examples

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example 1

Cloning of Mouse GFRα3

[0276] Using sequences from the neurturin receptor, now known as GFRα2 (“glial-cell-line-derived neurotrophic factor family receptor alpha”), a novel, potential member of the GFRα family was identified as a mouse EST in a public gene database (Accession Numbers W99197 (SEQ ID NO: 1), AA041935 (SEQ ID NO: 2) and AA050083 (SEQ ID NO: 3)). A DNA fragment corresponding to this potentially new receptor was obtained by Marathon PCR using mouse E15 cDNA (Clontech, Inc. USA) as template and PCR primers derived from the mouse EST. The PCR product was then used to screen a lambda gt10 mouse E15 library (Clontech, Inc. USA) to obtain a full length clone. The nucleotide sequence of the full length mouse cDNA is provided as SEQ ID NO: 4 (FIG. 1A-1B). The protein sequence (SEQ ID NO: 5; see FIG. 1A-1B) encoded by the isolated DNA was designated GFRα3, since it was determined to be a novel protein with sequence identity to GFRα1 (formerly the GDNF Receptor alpha) and GFRα2 ...

example 2

Isolation of cDNA Clones Encoding Human GFRα3

[0278] To identify rapidly whether a human homolog of this new receptor might exist, an expressed sequence tag (EST) DNA database (a proprietary EST database, LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST (INC3574209) was identified having the sequence:

(SEQ ID NO: 10)GCGCTGNNTGNCNGNANGNGGGGGCGGGAGGTGCCGGTCGAGGGAGCCCCGCTCTCAGAGCTCCAGGGGAGGAGCGANGGGAGCGCGGAGCCCGGCCGCCTACAGCTCGCCATGGTGCGCCCCCTGAACCCGCGACCGCTGCCGCCCGTAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCTCTCGCAGCCGGAGACCCCCTTCCCACAGAAAGCCGACTCATGAACAGCTGTCTCCAGGCCAGGAGGAAGTGCCAGGCTGATCCCACCTGC.

This sequence had 61% identity to the murine GFRα3.

[0279] To clone the corresponding full length cDNA, a panel of cDNA libraries were screened with primers:

newa3.F5′ GCC TCT CGC AGC CGG AGA CC 3′(SEQ ID NO: 11)newa3.R5′ CAG GTG GGA TCA GCC TGG CAC 3′(SEQ ID NO: 12)

DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protoco...

example 3

Use of GFRα3 as a Hybridization Probe

[0286] The following method describes use of a nucleotide sequence encoding GFRα3 as a hybridization probe.

[0287] DNA comprising the coding sequence of GFRα3 (shown in SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 16), or a fragment thereof, is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring GFRα3 or variants of GFRα3) in human tissue cDNA libraries, human tissue genomic libraries, RNA isolated from tissues, tissue preparations in situ, or chromosome preparations (such as for chromosome mapping).

[0288] Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled GFRα3-derived probe to the filters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filte...

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Abstract

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, vectors and host cells expressing, and immunoadhesions and antibodies to mammalian GFRα3, a novel α-subunit receptor of the GDNF (i.e. GFR) receptor family. It further relates to an assay for measuring activation of an α-subunit receptor by detecting tyrosine kinase receptor activation (i.e., autophosphorylation) or other activities related to ligand-induced α-subunit receptor homo-dimerization or homo-oligomerization.

Description

TECHNICAL FIELD [0001] The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides which are characterized by the presence of GFRα3 sequences, an α-subunit receptor. It further relates to an assay for measuring ligand-induced activation of an α-subunit receptor by detecting autophosphorylation of a kinase domain of an α-receptor-receptor protein tyrosine kinase (rPTK) fusion using a kinase receptor activation, enzyme-linked immunosorbent assay (KIRA ELISA) or by other means to detect α-subunit homodimerization. INTRODUCTION BACKGROUND [0002] Neurotrophic factors such as insulin-like growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, -4 / 5 and -6, ciliary neurotrophic factor, GDNF, and neurturin have been proposed as potential means for enhancing specific neuronal cell survival, for example, as a treatment for neurodegenerative diseases such as amyotrophic lateral ...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61K45/00A61P1/02A61P1/10G01N33/68A61P11/06A61P17/02A61P25/02A61P25/06A61P27/02C07K14/71C07K16/28C07K19/00C12N1/19C12N1/21C12N5/10C12N15/09C12N15/12C12Q1/42G01N33/573
CPCC07K14/71C07K16/2863C07K2319/00C12Q1/485G01N33/573G01N33/74G01N2500/04G01N2500/10C07K2317/75A61P1/02A61P1/10A61P11/06A61P17/02A61P19/02A61P25/00A61P25/02A61P25/06A61P27/02A61P29/00A61P9/00A61P9/12
Inventor DE SAUVAGE, FREDERICKLEIN, ROBERTPHILLIPS, HEIDIROSENTHAL, ARNON
Owner DE SAUVAGE FREDERIC
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