Constitutively Translocating Cell Line
The present invention relates to agonist-independent methods of screening for compounds that alter GPCR desensitization. Included in the present invention are cell lines containing GRKs, in which GPCRs are desensitized in the absence of agonist; the GRKs may be modified. The present invention relates to methods to determine if a GPCR is expressed at the plasma membrane, and if the GPCR has an affinity for arrestin. Modified GPCRs which have increased arrestin affinity are included in the present invention. These modified GPCRs are useful in methods to screen for compounds that alter desensitization, including both the agonist-independent methods and agonist-dependent methods described herein.
1 - 29 . (canceled)
30 . A host cell comprising a GPCR and a modified GRK.
31 . The host cell of claim 30 , wherein the GRK is inducible or over-expressed.
32 . The host cell of claim 30 , wherein said host cell further comprises arrestin, wherein said arrestin may be detectably labeled.
33 . The host cell of claim 30 , wherein at least one of the GPCR, GRK, another molecule involved in desensitization, or a molecule that interacts with a molecule involved in desensitization is detectably labeled.
34 . A method of modifying a nucleic acid encoding a GRK in which a GPCR is constitutively internalized, comprising:
(c) providing a nucleic acid encoding a GRK;
(b) mutating the nucleic acid encoding a GCRK such that the encoded GRK comprises a CAAX motif, wherein said modified GRK phosphorylates a GPCR in the absence of agonist; and
(c) expressing the modified GRK in a cell.
35 . The method of claim 34 , wherein the nucleic acid encoding a GRK comprises SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
36 . A kit for identifying a compound that modulates the internalization of a GPCR, comprising the host cell of claim 30 .
37 - 53 . (canceled)