Method of screening for modulators of HIV infection
The present invention provides polynucleotides that encode the chemokine receptors 88-2B or 88C and materials and methods for the recombinant production of these two chemokine receptors. Also provided are assays utilizing the polynucleotides which facilitate the identification of ligands and modulators of the chemokine receptors. Receptor fragments, ligands, modulators, and antibodies are useful in the detection and treatment of disease states associated with the chemokine receptors such as atherosclerosis, rheumatoid arthritis, tumor growth suppression, asthma, viral infection, AIDS, and other inflammatory conditions.
1. A method of screening for a modulator of 88C receptor activity, comprising the steps of:
(a) contacting in vitro a composition comprising an 88C receptor with a putative ligand, wherein the 88C receptor comprises the amino acid sequence of SEQ ID NO: 2; and
(b) detecting binding of the 88C receptor and the putative ligand;
wherein binding of the 88C receptor with the putative ligand is indicative of the 88C receptor having binding activity for the ligand.
2. The method according to claim 1 further comprising the step of screening a 88C ligand for the ability to affect 88C signal transduction activity, wherein an increase in 88C signal transduction activity indicates the ligand is an activator, or a decrease in 88C signal transduction activity indicates the ligand is an inhibitor.
3. A method for identifying a ligand capable of interacting with the chemokine receptor 88C that comprises (i) SEQ ID NO: 2 or (ii) an extracellular domain having residues 89-112 of SEQ ID NO: 2 and which have biological activity of chemokine receptor 88c, said method comprises the steps of:
(a) providing a host cell expressing said receptor or a membrane preparation comprising said receptor,
(b) contacting and incubating said receptor with a ligand, and
(c) identifying the presence of a ligand that specifically interacts with said receptor.
4. The method according to claim 3 , wherein said fragment further comprises the amino acid sequence spanning amino acids 1-32, 56-67, 125-145, 166-191, 213-235, 259-280 or 301-352 of SEQ ID NO: 2.
5. The method according to claim 3 , wherein said ligand is labeled.
6. The method according to claim 3 , wherein said ligand is an agonist or an antagonist.
7. The method according to claim 3 , wherein said interaction is identified by measuring the concentration or level of activity of a component of a signal transduction pathway.
8. The method according to claim 7 , wherein said signal transduction pathway is identified by measuring an activity selected from the group consisting of: calcium concentration, IP3 concentration, GTP hydrolysis and phospholipase C activity.
9. The method according to any claim 3 , wherein said receptor or said fragment is contacted and incubated with a ligand and a further test compound.
10. The method according to claim 3 , wherein said cell or said cell membrane preparation are selected from the group consisting of CHO-KI, HEK293, BHK21 and COS-7.