A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Abstract

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533–555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533–555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533–555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533–555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533–555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested. Synthetic peptide amides lacking structural criteria associated with G protein activation did not affect guanine nucleotide exchange or FSH-stimulated estradiol levels, even at concentrations significantly higher than used for the receptor-related peptide. Our results are consistent with peptide interaction with signal-transducing G protein. These data suggest that the 3i loop peptide acts as an antagonist of FSH receptor-mediated G protein activation, and inhibits FSH receptor-mediated signal transduction in intact Sertoli cells. The ability of this FSH receptor-related peptide to affect intracellular processes in intact cultured rat Sertoli cells through its apparent interaction with G protein suggests a novel approach for control of FSH action.