Constitutively translocating cell line
View Patent ↗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. A method of identifying a compound which alters G protein-coupled receptor (GPCR) internalization, comprising:
a) providing a cell comprising a CPCR, an arrestin, and a modified G protein-coupled receptor kinase (GRK) comprising one or more modifications in the amino acid sequence of the four C-terminal amino acids of the GRK as compared to a wild type GRK, wherein said GPCR is at least partially internalized in an agonist-independent manner upon expression of said modified GRK;
b) exposing said cell to the compound(s);
c) determining the cellular distribution of the GPCR, arrestin, or modified GRK, wherein said modified GRK alters GPCR desensitization in the absence of an agonist as compared to a wild type GRK; and
d) monitoring a difference between (1) the distribution of the GPCR, arrestin, or modified GRK in the cell in the presence of the compound(s) and (2) the distribution of the GPCR, arrestin, or modified GRK in the cell in the absence of the compound(s).
2. The method of claim 1 , wherein the expression of the modified GRK of step (a) is inducible.
3. The method of claim 1 , wherein the modified GRK comprises a CAAX (SEQ ID NO:95) motif, wherein C is cysteine, A is an aliphatic amino acid, and X is the wild type C-terminal amino acid of GRK.
4. The method of claim 1 , wherein the GPCR comprises one or more modifications in the amino acid sequence of its carboxy-terminal tail, to have enhanced phosphorylation by the modified GRK as compared to a wild type GPCR.
5. The method of claim 1 , wherein the CPCR is (β 2 AR(Y326A).
6. The method of claim 1 , wherein the GPCR is a GPCR listed in FIG. 1 , an orphan GPCR, a taste receptor, a Class A GPCR, a Class B GPCR, a mutant GPCR, or a biologically active fragment thereof.
7. The method of claim 1 , wherein the modified or wild type GRK is GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, or a biologically active fragment thereof.
8. The method of claim 1 , wherein the GPCR, GRK, or arrestin is detectably labeled.
9. The method of claim 1 , wherein a molecule involved in GPCR desensitization is detectably labeled, or a molecule that interacts with a molecule involved in GPCR desensitization is detectably labeled.
10. The method of claim 1 , wherein the arrestin is visual arrestin, cone arrestin, β-arrestin 1, β-arrestin 2, or a biologically active fragment thereof.
11. The method of claim 1 , wherein an agonist of said GPCR is not provided.
12. The method of claim 1 , wherein a difference between (1) and (2) of step (d) indicates modulation of GPCR internalization.
13. The method of claim 1 , wherein the GPCR is AT1AR.
14. The method of claim 3 , wherein said CAAX motif is CVLL (SEQ ID NO:94).