Gfralpha3 and its uses

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.

1 .- 25 . (canceled) 
     
     
         26 . A method for measuring agonist binding to a polypeptide comprising an agonist-binding domain of an α-subunit receptor, comprising the steps of exposing the polypeptide positioned in a cell membrane to a candidate agonist and measuring homo-dimerization or homo-oligomerization of the polypeptide. 
     
     
         27 . The method of  claim 26 , wherein the α-subunit receptor is a GFRα-receptor. 
     
     
         28 . The method of  claim 26 , wherein the polypeptide further comprises a dimerization- or oligomerization-activated enzymatic domain and homo-dimerization or homo-oligomerization is detected by measuring enzymatic activity of the polypeptide. 
     
     
         29 . The method of  claim 28 , wherein the enzymatic domain is the intracellular autocatalytic domain of a receptor tyrosine kinase and homo-dimerization or homo-oligomerization is detected by measuring autophosphorylation of the polypeptide. 
     
     
         30 . A method of measuring autophosphorylation of a polypeptide receptor construct comprising a ligand-binding domain of an α-subunit receptor, the intracellular catalytic domain of a tyrosine kinase receptor, and a flag epitope, comprising the steps of:
 (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein, positioned in their membranes, the cells have the polypeptide receptor construct;   (b) exposing the adhering cells to an analyte;   (c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;   (d) coating a second solid phase with a capture agent which binds specifically to the flag epitope so that the capture agent adheres to the second solid phase;   (e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the receptor construct adheres to the second solid phase;   (f) washing the second solid phase so as to remove unbound cell lysate;   (g) exposing the adhering receptor construct to an anti-phosphotyrosine antibody which identifies phosphorylated tyrosine residues in the tyrosine kinase receptor; and   (h) measuring binding of the anti-phosphotyrosine antibody to the adhering receptor construct.   
     
     
         31 - 40 . (canceled) 
     
     
         41 . The method of  claim 30  wherein the α-subunit receptor is a GFRα-receptor. 
     
     
         42 . The method of  claim 30  wherein the tvrosine kinase is a Rse receptor, a trkA receptor, a trk B receptor, or a trkC receptor, and the receptor construct further comprises the transmembrane domain of the Rse receptor and the flag epitope comprises the gD polypeptide. 
     
     
         43 . The method of  claim 30  wherein the analyte comprises an agonist for the α-subunit receptor. 
     
     
         44 . The method of  claim 30  wherein the analyte comprises an antagonist for the α-subunit receptor. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 30  wherein the analyte is a composition which comprises an antagonist and an agonist for the α-subunit receptor and the assay measures the ability of the antagonist to bind to the agonist and thereby reduce activation of the polypeptide construct by the agonist. 
     
     
         47 . A method for measuring autophosphorylation of a polypeptide receptor construct comprising a ligand-binding domain of an α-subunit receptor, the intracellular catalytic domain of a tyrosine kinase receptor, and a flag epitope, comprising the steps of:
 (a) coating a well of a first assay plate with a homogeneous population of adherent cells so that the cells adhere to the well, wherein the cells have the polypeptide receptor construct positioned in the cell membranes thereof;   (b) exposing the adhering cells to an analyte;   (c) solubilizing the adhering cells thereby releasing cell lysate therefrom;   (d) coating a well of a second assay plate with a capture agent which binds specifically to the polypeptide receptor construct so that the capture agent adheres to the well;   (e) exposing the cell lysate obtained in step (c) to the adhering capture agent so that the polypeptide receptor construct adheres to the well;   (f) washing the well so as to remove unbound cell lysate;   (g) exposing the adhering polypeptide receptor construct to an anti-phosphotyrosine antibody which binds selectively to phosphorylated tyrosine residues in the polypeptide receptor construct;   (h) measuring binding of the anti-phosphotyrosine antibody to the adhering polypeptide receptor construct.   
     
     
         48 . The method of  claim 47  wherein the α-subunit receptor is a GFRα-receptor. 
     
     
         49 . A polypeptide comprising a GFRα receptor ligand-binding domain, a flag polypeptide, and an intracellular catalytic domain of a tyrosine kinase receptor. 
     
     
         50 - 66 . (canceled) 
     
     
         67 . An assay for measuring phosphorylation of polypeptide receptor construct comprising a ligand-binding domain of an α-subunit receptor, the intracellular catalytic domain of a kinase receptor, and a flag epitope comprising the steps of:
 (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein the cells comprise the polypeptide receptor construct;   (b) exposing the adhering cells to an analyte;   (c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;   (d) coating a second solid phase with a capture agent which binds specifically to the flag polypeptide so that the capture agent adheres to the second solid phase:   (e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the receptor construct adheres to the second solid phase;   (f) washing the second solid phase, so as to remove unbound cell lysate;   (g) exposing the adhering kinase construct to an antibody which identifies phosphorylated residues in the receptor construct; and   (h) measuring binding of the antibody to the adhering receptor construct.   
     
     
         68 . The assay of  claim 67  wherein the or α-receptor is a GFRα-receptor. 
     
     
         69 . A kit comprising a solid phase coated with a capture agent which binds-specifically to a flag polypeptide, and a polypeptide comprising an α-subunit receptor ligand-binding domain, a flag polypeptide, and an intracellular catalytic domain of a tyrosine kinase receptor. 
     
     
         70 . A method of treating a subject in need of treatment for a condition selected from pain, inflammation, dysfunction of the peripheral nervous system, and dysfunction of the autonomic nervous system, comprising administering to said subject an effective amount of a nucleic acid molecule encoding a GFRα3 receptor comprising the polypeptide of SEQ ID NO: 17 or an effective amount of a GFRα3 receptor comprising the polypeptide of SEQ ID NO: 17. 
     
     
         71 . The method of  claim 71 , wherein said condition selected from pain, inflammation, dysfunction of the peripheral nervous system, and dysfunction of the autonomic nervous system, comprises a condition selected from neuropathic pain, non-neuropathic pain, chronic pain, chronic back pain, peripheral neuropathy, a disturbance in blood pressure, a disturbance in cardiac rhythm, a gastrointestinal dysfunction, impotence, urinary incontinence, post-herpetic neuralgia, shingles, asthma, irritable bowel, inflammatory bowel, cystitis, headache, migraine, arthritis, spinal cord injury, constipation, hypertension, mucositis, dry mouth, dry eyes, fibromyalgia, and wound healing.