The agonist-induced phosphorylation of the rat follitropin receptor maps to the first and third intracellular loops

Previous results from this laboratory have shown that the rat FSH receptor (rFSHR) becomes phosphorylated on S/T residues upon stimulation of transfected cells with human (h)FSH and that a truncation of the C-terminal tail that removes 12 of the 25 intracellular S/T residues does not affect phosphorylation. Based on the results of phosphopeptide-mapping experiments we analyzed three new mutants. rFSHR-1L and rFSHR-3L were constructed by mutating the S/T residues in the first intracellular loop or the third intracellular loop, respectively. rFSHR-(3L+CT) was constructed by mutating all the S/T residues in the third loop as well as S-624, the only C-terminal tail residue that was not previously eliminated as a potential phosphorylation site. All mutants were biologically active. The agonist-induced phosphorylation of rFSHR-3L and rFSHR-(3L+CT) were partially reduced, while that of rFSHR-1L was almost completely lost. The agonist-induced uncoupling of rFSHR-1L and rFSHR-3L are retarded to about the same extent, while the agonist-induced internalization is retarded only in rFSHR-1L. Four major conclusions can be made from the present studies: 1) the phosphorylated rFSHR is a common molecular intermediate in agonist-induced uncoupling and internalization; 2) agonist-induced phosphorylation of the rFSHR maps to the first and third intracellular loops; 3) the phosphorylation of the third intracellular loop facilitates agonist-induced uncoupling but is not necessary for agonist-induced internalization; 4) agonist-induced internalization is facilitated by phosphorylation but it is not known if only the first loop, only the third loop, or both the first and third loops need to be phosphorylated for this response.