Gonadotropin-releasing hormone and thyrotropin-releasing hormone regulation of g protein function in the rat anterior pituitary lobe.
Abstract In order to evaluate the role of guanine nucleotide-dependent transducer proteins (G proteins) in hormone-mediated signal transduction in the anterior pituitary lobe, we examined the effect of gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) on two parameters of G protein function, namely [(35) S]GTP(gamma)S binding and low K(m)GTPase activity. Plasma membranes were prepared from anterior pituitary lobes of adult male rats using conventional procedures. GTP binding was determined by incubating 2 to 5 mug membrane protein with approximately 100,000 cpm [(35) S]GTP(gamma)S in a buffer containing 20 mM Tris- HCl, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCI at a pH of 7.4 for 10 or 15 min at 37 degrees C GnRH agonist and TRH stimulated high affinity [(35) S]GTP(gamma)S binding in a concentration-dependent manner. GTP binding was maximally stimulated by GnRH agonist (1 muM) and TRH (0.1 muM) by up to 27% and 34%, respectively. A time-course study revealed that 1 muM GnRH agonist stimulated GTP binding by 30% at 15 min; 0.1 muM TRH stimulated GTP binding by 23% at 1 min, 18% at 5 min and 25% at 10 min. A stable GTP analog, 5'-guanylylimidodiphosphate, inhibited GnRH- as well as TRH-stimulated GTP binding. GnRH antagonist did not affect GTP binding. However, in the presence of the antagonist, stimulation of GTP binding by the GnRH agonist was completely blocked. The low K(m)GTPase activity (EC 3.6.1.-), another parameter of G protein function, was assayed in 2 to 5 mug membrane protein using [gamma-(32) P]GTP at 37 degrees C in an ATP-regenerating buffer containing 1 muM unlabeled GTP. GnRH agonist (0.1 muM) and TRH (1 muM) maximally stimulated this GTPase activity by up to 50% and 40%, respectively. GnRH agonist (1 muM) stimulated the GTPase activity by 30% at 10 min and 48% at 30 min. TRH (1 muM) stimulated the GTPase activity at all time points monitored; stimulation was 46% at 5 min, 49% at 20 min, and 41% at 30 min. Interestingly, the GnRH antagonist stimulated GTPase activity by about 20%, but inhibited GnRH agonist-stimulated GTPase activity in a concentration-dependent manner. These results indicate that the binding of GnRH and TRH to their receptors results in interaction of the receptor with a G protein and activation of the G protein cycle.
Ravindra
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Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2300, USA.
Aronstam
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