The effects of aprotinin on follicle-stimulating hormone binding and signal transduction in bovine calf testis.

Aprotinin (bovine pancreatic trypsin inhibitor), a serine protease inhibitor, caused a dose-dependent inhibition of [125I]human FSH ([ 125I]hFSH) binding to 1) an FSH receptor-enriched light membrane fraction prepared from bovine calf testes homogenates, 2) Triton X-100-solubilized FSH receptor, and 3) proteoliposomes containing incorporated FSH receptor-G-protein-adenylate cyclase (AC) complexes. Equilibrium binding studies with the solubilized receptor indicated that the effect of aprotinin on [125I]hFSH binding was due to a decrease in the Ka of the receptor, with no change in FSH-binding capacity. The rate of association of [125I]hFSH with its receptor was reduced by 50% in the presence of aprotinin, but no effect on dissociation of FSH-receptor complexes was evident. Aprotinin, at a concentration (250 microM) that inhibited binding of [125I]hFSH to the membrane receptor by 25%, completely inhibited basal, fluoride-stimulated and FSH-stimulated AC activity. However, aprotinin, at a concentration (50 microM) that had little effect on [125I]hFSH binding, markedly enhanced basal AC activity (3.4-fold) to the level of fluoride and FSH stimulation. Aprotinin did not inhibit [3H]5'-guanylylimidodiphosphate binding to FSH receptor-enriched membranes, suggesting that its effects on the affinity of the receptor for FSH and on AC activation were not mediated through an interaction with FSH receptor-associated G-protein. No serine protease activity could be detected in any of the receptor or hormone preparations used in this study. The ability of aprotinin to inhibit binding of [125I]hFSH to the Triton X-100-solubilized receptor and to the soluble receptor incorporated into proteoliposomes as well as to the FSH receptor-enriched membrane fraction, all of which are free of serine protease activity, is consistent with the notion that aprotinin may directly interact with the FSH receptor to sterically hinder binding of FSH. Furthermore, the apparent direct effect of aprotinin on basal as well as FSH-stimulated AC activity suggests its general usefulness in studies on the mechanism of signal transduction for ligands thought to operate via the cAMP second messenger system.