Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agoni st-induced phosphorylation and internalization of the rat FSH receptor (rFS HR). Western blots of human kidney 293 cells (the cell line used in transfe ction experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterp arts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GR K4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK 4 alpha, or GRK6 resulted in an increase in the agonist-induced phosphoryla tion of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enh anced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and int ernalization, we inhibited endogenous GRK2 by overexpression of a kinase-de ficient mutant of GRK2 or G alpha t, a scavenger of G beta gamma. We also i nhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, bu t only the kinase-deficient mutant of GRK2 and G alpha t inhibited internal ization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internaliza tion, suggests that different GRKs have differential effects on receptor in ternalization.