Guanosine diphosphate binding, metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Abstract

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [ 3H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K a of 4.5 ·10 6M −1 and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′- O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [ 3H]GDP binding (ED 50 = 4.8·10 −8M), whereas guanosine 5′- O-[2-thio]diphosphate (Gpp[S]) was less potent (ED 50 = 3.4·10 −7M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg 2+ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited ( P < 0.05) Gppp[S]-stimulated adenylate cyclase activity ( K i = 1.8·10 −7M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K off value of 6 ± 1·min −1. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.