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Glial cell line-derived neurotrophic factor receptor

a neurotrophic factor and receptor technology, applied in the direction of dna/rna fragmentation, peptide/protein ingredient, depsipeptide, etc., can solve the problems of not being able to survive in culture medium, and the mode of action of gdnf in vivo is not clearly elucidated

Inactive Publication Date: 2003-09-18
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032] Additional aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following description, which details the practice of the present invention.

Problems solved by technology

The mode of action of GDNF in vivo is not clearly elucidated in the art, in part due to the absence of information on a receptor for GDNF.
Host cells that were not transformed with the vector will not contain the selection gene, and therefore, they will not survive in the culture medium.

Method used

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  • Glial cell line-derived neurotrophic factor receptor
  • Glial cell line-derived neurotrophic factor receptor
  • Glial cell line-derived neurotrophic factor receptor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Identification of Cells Expressing High Affinity GDNF Binding Sites

[0217] Expression cloning involved the selection of a source of mRNA which is likely to contain significant levels of the target transcript. Retina photoreceptor cells were identified as responsive to GDNF at very low concentrations, suggesting the existence of a functional, high affinity receptor. To confirm that rat photoreceptor cells did express a high affinity receptor for GDNF, [.sup.125I]GDNF binding and photographic emulsion analysis were carried out.

[0218] Rat Retinal Cell Cultures

[0219] The neural retinas of 5-day-old C57B1 / 6 mouse pups or 3-day-old Sprague-Dawley rat pups (Jackson Laboratories, Bar Harbor, Mass.) were carefully removed and dissected free of the pigment epithelium, cut into 1 mm.sup.2 fragments and placed into ice-cold phosphate-buffered saline (PBS). The retinas were then transferred into 10 mL of Hank's balanced salt solution (HBSS) containing 120 units papain and 2000 units DNAase and in...

example 2

Expression Cloning of a GDNFR from Photoreceptor Cells

[0227] Rat photoreceptor cells were selected as a possible source of a high affinity receptor for GDNF based upon their cell surface binding of radiolabeled GDNF and their ability to respond to very low concentrations of the ligand, as described in Example 1. In order to identify the receptor, a size-selected cDNA library of approximately 50,000 independent clones was constructed using a mammalian expression vector (a derivative of pSR, Takebe et al., 1988 supra) and mRNA isolated from cultured post-natal rat photoreceptor cells, by the methods described below. The library was divided into pools of approximately 1,500 to 2,000 independent clones and screened using an established expression cloning approach (Gearing et al., EMBO Journal, 8, 3667-3676, 1989). Plasmid DNA representing each pool of the library was prepared and transfected into COS7 cells grown on plastic microscope slide flaskettes (Nunc, Naperville, Ill.).

[0228] The...

example 3

DNA Sequencing and Sequence Analysis

[0237] DNA from positive, single clones was prepared and sequenced using an automated ABI373A DNA sequencer (Perkin / Elmer Applied Biosystems, Santa Clara, Calif. ) and dideoxy-dye-terminators, according to manufacturer's instructions. Comparison of GDNF receptor sequence with all available public databases was performed using the FASTA (Pearson and Lipman, Proceedings Of The National Academy Of Sciences U.S.A., 85, 2444-2448, 1988) program algorithm as described in the University of Wisconsin Genetics Computer Group package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, Madison, Wis.).

[0238] Sequence Characterization of the Rat GDNFR

[0239] Plasmid DNA from the clones described in Example 2, above, was prepared and submitted for DNA sequence analysis. Nucleotide sequence analysis of the cloned 2138 bp rat cDNA revealed a single large open reading frame encoding a translation protein of 468 amino acid ...

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Abstract

The present invention relates to glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophin that exhibits a broad spectrum of biological activities on a variety of cell types from both the central and peripheral nervous systems. The present invention involves the cloning and characterization of a high affinity receptor for GDNF. This molecule has been named GDNF receptor (GDNFR) since it is the first known component of a receptor system. Nucleic acid and amino acid sequences are described for GDNFR protein products. A hydrophobic domain with the features of a signal peptide is found at the amino terminus, while a second hydrophobic domain at the carboxy terminus is involved in the linkage of the receptor to the cell membrane. The lack of a transmembrane domain and cytoplasmic region indicates that GDNFR requires one or more accessory molecules in order to mediate transmembrane signaling. GDNFR mRNA is widely distributed in both nervous system and non-neural tissues, consistent with the similar distribution found for GDNF.

Description

[0001] This application is a continuation of U.S. application Ser. No. 08 / 837,199, filed Apr. 14, 1997, now pending, which claims the benefit of U.S. provisional application serial No. 60 / 015,907, filed April 22, 1996 and U.S. provisional application serial No. 60 / 017,221, filed May 9, 1996, which are incorporated by reference herein.1. FIELD OF THE INVENTION[0002] The present invention relates to receptors for glial cell line-derived neurotrophic factor (GDNF) and provides nucleic acid and amino acid sequences encoding GDNF receptor (GDNFR). The present invention also relates to therapeutic techniques for the treatment of GDNF-responsive conditions.2. BACKGROUND OF THE INVENTION[0003] Glial Cell Line-Derived Neurotrophic Factor[0004] Glial cell line-derived neurotrophic factor (GDNF) was initially isolated and cloned from rat B49 cells as a potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons (Lin et al., Science, 260, 1130-1132, 1993). Recent studies ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/48A61K31/00A61K38/00A61K38/17A61K39/395C12N15/09A61P25/16A61P25/28C07K14/705C07K14/71C07K16/28C12N1/21C12N5/10C12N15/12C12P21/02C12P21/08G01N33/53G01N33/566G01N33/68
CPCA61K38/00C07K14/715C07K14/71A61P25/16A61P25/28C12N15/11C12N1/20C12N5/10G01N33/68C07K16/28A61K38/17A61K9/48
Inventor FOX, GARY M.JING, SHUQIANWEN, DUANZHI
Owner AMGEN INC
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