TRUNCATION OF THE C-TERMINAL TAIL OF THE FOLLITROPIN RECEPTOR DOES NOT IMPAIR THE AGONIST-INDUCED OR PHORBOL ESTER-INDUCED RECEPTOR PHOSPHORYLATION AND UNCOUPLING

We have recently shown that addition of follitropin (FSH) or a phorbol ester (phorbol la-myristate 13-acetate (PMA)) to cells expressing the recombinant follitropin receptor (FSHR) results in both phosphorylati on and uncoupling of the FSHR from adenylyl cyclase, In the light of f indings reported with other G protein-coupled receptors we have propos ed that phosphorylation of the FSHR mediates the uncoupling from adeny lyl cyclase. The experiments described herein represent the first atte mpt to determine the location of the amino acid residues that become p hosphorylated in FSHR and to test the hypothesis that phosphorylation is responsible for uncoupling of FSHR from adenylyl cyclase. As a firs t step in identifying which residues may be phosphorylated in response to hFSH and PMA, we constructed a mutant of the FSHR cDNA in which th e C-terminal cytoplasmic tail was truncated at residue 635 (FSHR-t635) , thus removing all but one of the potential phosphorylation sites pre sent in the C-terminal tail, Cells expressing FSHR-t635 bind hFSH with the appropriate affinity and respond with increases in cAMP and inosi tol phosphate accumulation. The maximal cAMP and inositol phosphate re sponses of cells expressing FSHR-t635 are higher than those of cells e xpressing the wild type FSHR, but the concentration of hFSH required t o elicit these responses is similar in both cell lines, Immunoprecipit ation of FSHR-t635 shows that the truncated receptor is still effectiv ely phosphorylated in response to hFSH or PMA. Phosphoamino acid analy sis reveals that, like the wild-type FSHR, FSHR-t635 phosphorylation o ccurs on serine and threonine residues, Peptide mapping suggests that the phosphorylated residues in the FSHR and FSHR-t635 are located with in the same areas of the intracellular regions of the receptors. In ad dition to stimulating phosphorylation of FSHR-t635, hFSH and PMA also effectively uncouple the truncated receptor from adenylyl cyclase, Tak en together, these data show that hFSH and PMA can both phosphorylate and uncouple a FSH receptor species with a cytoplasmic tail truncated at residue 635.