The association of arrestin-3 with the follitropin receptor depends on receptor activation and phosphorylation

Abstract

We have recently shown that the binding of arrestin-3 to the lutropin receptor (LHR) is dependent mostly on receptor activation rather than on phosphorylation. The experiments presented here were designed to test the involvement of these two events in the association of arrestin-3 with the closely related follitropin receptor (FSHR). Activation of the FSHR leads to the phosphorylation of residues in the first and third intracellular loops. Mutation of the phosphorylation sites in the third intracellular loop of the rat (r) FSHR partially reduces phosphorylation but has no effect on arrestin-3 association. Mutation of the phosphorylation sites in the first intracellular loop abolishes phosphorylation and arrestin-3 association. Dominant-negative mutants of G protein-coupled receptor kinase (GRKs) 2 and 6 inhibit rFSHR phosphorylation to the same extent but only the dominant-negative mutant of GRK2 inhibits arrestin-3 association. Two mutations of the rFSHR (D389N and Y530F) that impair activation and abolish phosphorylation also impair arrestin-3 binding. GRK2 restores the phosphorylation of both mutants but it restores arrestin-3 association only to the D389N mutant. We conclude that, in contrast to the data obtained with the LHR, the association of arrestin-3 with the FSHR is dependent on receptor phosphorylation. The phosphorylation of the third intracellular loop residues is not needed for arrestin-3 association, however.