Wild-type receptor assays
View Patent ↗A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of β-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of β-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the β-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.
1. A method for determining activation of a cell surface receptor that binds to an intracellular binding partner and endocytoses with the intracellular binding partner to associate with an endosome, comprising the steps of:
(a) providing a cell expressing (i) a fusion protein of a fragment of β-galactosidase fused to the intracellular binding partner, (ii) a fusion protein of a fragment of β-galactosidase fused to an endosome-associated protein portion, wherein said fragments of β-galactosidase weakly bind each other so that complementation activity depends on interaction of the fusion proteins, and (iii) the cell surface receptor, wherein the cell surface receptor is unlabeled;
(b) contacting said cell with a ligand for sufficient time for said ligand to bind to said receptor and any binding of said intracellular binding partner to said receptor to occur; then
(c) lysing said cells with a lysing medium comprising a substrate for said β-galactosidase that produces a detectable product; and
(d) determining said detectable product, wherein production of said detectable product indicates endosomal localization of said receptor.
2. A method according to claim 1 , wherein said endosome-associated protein is a fragment of endofin binding to the endosome.
3. A method according to claim 1 , wherein said cell is a CHO cell.
4. A method according to claim 1 , wherein said receptor is phosphorylated upon activation and said binding partner is a protein that comprises a domain that specifically binds to the phosphorylated form of the cell surface receptor.
5. A method according to claim 1 , wherein said fragment fused to said endosome-associated protein is a fragment of from 36 to 60 amino acids.
6. The method of claim 1 wherein said cell surface receptor and said intracellular binding partner are selected from the group consisting of GPCR and arrestin, tyrosine kinase and Hrs protein.
7. A method for determining activation by a ligand of an unlabeled GPCR, which binds to arrestin upon activation and endocytoses to associate with an endosome, said method comprising the steps of:
(a) providing cells expressing (i) a fusion protein of a fragment of β-galactosidase with arrestin, (ii) a complementary fragment of β-galactosidase with an endosome-associated protein, and (iii) said unlabeled GPCR, wherein said complementary fragments weakly bind to each other so that complementation activity depends on interaction of the fusion proteins;
(b) contacting said cells with said ligand for sufficient time for said ligand to bind to said GPCR and any resultant binding of arrestin to said GPCR to occur;
(c) lysing said cells with a lysing medium comprising a substrate for said β-galactosidase that produces a detectable product; and
(d) determining said detectable product, wherein production of said detectable product indicates activation of said GPCR.
8. A method according to claim 7 , wherein said endosome-associated protein comprises the FYVE domain of endofin.
9. A method according to claim 7 , wherein said cell is a CHO cell.
10. A method according to claim 7 , wherein said GPCR is Type B.
11. A method according to claim 7 , wherein said fragment fused to said endosome-associated protein is a fragment of from 36 to 60 amino acids.
12. A method according to claim 7 , wherein said GPCR is SSTR2.
13. A kit for use in GPCR translocation assays, comprising a genetic construct encoding arrestin fused to a fragment of β-galactosidase and a genetic construct encoding a complementary fragment of β-galactosidase fused to an endosome-associated protein.
14. The kit of claim 13 where the endosome-associated protein is endofin.
15. The kit of claim 13 further comprising a genetic construct encoding a GPCR.