Methods of identifying modulators of ChemerinR polypeptides
The present invention relates to a G-protein coupled receptor and a novel ligand therefor. The invention provides screening assays for the identification of candidate compounds which modulate the activity of the G-protein coupled receptor, as well as assays useful for the diagnosis and treatment of a disease or disorder related to the dysregulation of G-protein coupled receptor signaling.
1. A method of identifying an agent that modulates the function of ChemerinR polypeptide, wherein said ChemerinR polypeptide sequence is SEQ ID NO: 2, or 4, or 6, said method comprising:
contacting said ChemerinR polypeptide with a Chemerin polypeptide comprising an amino acid sequence selected from the group consisting of YFPGQFAFS (SEQ ID NO: 61) and FLPGQFAFS (SEQ ID NO: 47) in the presence or absence of a candidate modulator; and
measuring a signaling activity of said ChemerinR polypeptide, wherein a change in the activity in the presence of said candidate modulator relative to the activity in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the function of said ChemerinR polypeptide.
2. The method of claim 1 , wherein said ChemerinR polypeptide activity measured in the presence of said candidate modulator is reduced by at least 10% of the activity in the absence of said candidate modulator.
3. A method of identifying an agent that increases the signaling of a ChemerinR polypeptide, wherein said ChemerinR polypeptide sequence is SEQ ID NO: 2, or 4, or 6, said method comprising:
(a) contacting said ChemerinR polypeptide with a candidate modulator;
(b) measuring a signaling activity of said ChemerinR polypeptide in the presence of said candidate modulator; and
(c) comparing said activity measured in the presence of said candidate modulator to said activity measured in the absence of said candidate modulator in a reaction in which said ChemerinR polypeptide is contacted with a Chemerin polypeptide, said Chemerin polypeptide comprising an amino acid sequence selected from the group consisting of YFPGQFAFS (SEQ ID NO: 61) and FLPGQFAFS (SEQ ID NO: 47), wherein said candidate modulator is identified as an agent that increases the signaling of said ChemerinR polypeptide when the amount of said activity measured in the presence of said candidate modulator is at least 10% of the amount induced by said Chemerin polypeptide.
4. A method of identifying an agent that decreases the signaling of a ChemerinR polypeptide, wherein said ChemerinR polypeptide sequence is SEQ ID NO: 2, or 4, or 6, said method comprising:
(a) contacting said ChemerinR polypeptide with a Chemerin polypeptide comprising an amino acid sequence selected from the group consisting of YFPGQFAFS (SEQ ID NO: 61) and FLPGQFAFS (SEQ ID NO: 47) in the presence or absence of a candidate modulator;
(b) measuring a signaling activity of said ChemerinR polypeptide in the presence or absence of said candidate modulator; and
(c) comparing said activity measured in the presence of said candidate modulator to said activity measured in the absence of said candidate modulator, wherein a decrease in said activity in the presence of said candidate modulator relative to the activity in the absence of said candidate modulator identifies said candidate modulator as an agent that decreases the signaling of said ChemerinR polypeptide.
5. The method according to any one of claims 1 , 3 and 4 , wherein said candidate modulator is present in a sample.
6. The method according to any one of claims 1 , 3 and 4 , wherein said Chemerin polypeptide is detectably labeled.
7. The method according to any one of claims 1 , 3 and 4 , wherein said ChemerinR polypeptide is expressed in or on a cell.
8. The method according to any one of claims 1 , 3 and 4 , wherein said ChemerinR is present in a cell membrane.
9. The method according to any one of claims 1 , 3 and 4 , further performed in the presence of Galpha16.
10. The method according to any one of claims 1 , 3 and 4 , wherein said measuring is performed using a method selected from the group consisting of, label displacement, surface plasmon resonance, fluorescence resonance energy transfer, fluorescence quenching and fluorescence polarization.
11. The method according to any one of claims 1 , 3 and 4 , wherein said measuring the signaling activity of said ChemerinR polypeptide comprises detecting a change in the level of a second messenger.
12. The method according to claim 11 , wherein the step of measuring a signaling activity comprises measurement of guanine nucleotide binding or exchange, adenylate cyclase activity, cAMP, protein kinase C activity, phosphatidylinositol breakdown, diacylglycerol, inositol triphosphate, intracellular calcium, arachinoid acid concentration, MAP kinase activity, tyrosine kinase activity or reporter gene expression.
13. The method of claim 12 , wherein said measuring a signaling activity comprises using an aequorin-based assay.
14. The method according to any one of claims 1 , 3 and 4 , wherein said agent is selected from the group consisting of a peptide, a polypeptide, and antibody or antigen-binding fragment thereof, a lipid, a carbohydrate, a nucleic acid and a small organic molecule.
15. The method of any one of claims 1 , 3 and 4 , wherein said Chemerin polypeptide consists of the amino acid sequence of SEQ ID NO: 14.