THE AGONIST-INDUCED PHOSPHORYLATION OF THE RAT FOLLITROPIN RECEPTOR MAPS TO THE FIRST AND 3RD INTRACELLULAR LOOPS

Previous results from this laboratory have shown that the rat FSH rece ptor (rFSHR) becomes phosphorylated on S/T residues upon stimulation o f transfected cells with human (h)FSH and that a truncation of the C-t erminal tail that removes 12 of the 25 intracellular S/T residues does not affect phosphorylation. Based on the results of phosphopeptide-ma pping experiments we analyzed three new mutants. rFSHR-1L and rFSHR-3L were constructed by mutating the S/T residues in the first intracellu lar 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 previousl y eliminated as a potential phosphorylation site. All mutants were bio logically active. The agonist-induced phosphorylation of rFSHR-3L and rFSHR-(3L+CT) were partially reduced, while that of rFSHR-1L was almos t completely lost. The agonist-induced uncoupling of rFSHR-1L and rFSH R-3L are retarded to about the same extent, while the agonist-induced internalization is retarded only in rFSHR-1L. Four major conclusions c an be made from the present studies: 1) the phosphorylated rFSHR is a common molecular intermediate in agonist-induced uncoupling and intern alization; 2) agonist-induced phosphorylation of the rFSHR maps to the first and third intracellular loops; 3) the phosphorylation of the th ird intracellular loop facilitates agonist-induced uncoupling but is n ot necessary for agonist-induced internalization; 4) agonist-induced i nternalization is facilitated by phosphorylation but it is not known i f only the first loop, only the third loop, or both the first and thir d loops need to be phosphorylated for this response.