Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal an activation- and phosphorylation-independent but arrestin-dependent pathway for internalization.

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.