The role of membrane-associated tubulin in TRH receptor-G protein coupling was investigated with the use of compounds that influence tubulin function. TRH-stimulated G protein GTPase activity in GH3cell membranes was used to determine receptor-G protein coupling. TRH-stimulated GTPase activity was abolished by Gqαantibody, suggesting that TRH receptor coupling to Gqresults in the activation of Gqαand the subsequent hydrolysis of GTP. TRH (1 μM) stimulated the enzymatic activity by up to 69 pmol/min/mg protein, and in the presence of 1 μMcolchicine the hormone-stimulated activity was only 26 pmol/min/mg protein. Similar inhibition of TRH receptor-G protein coupling was observed with tubulin antibodies and purified tubulin, suggesting that perturbation of membrane-associated tubulin and/or tubulin-G protein interaction by these compounds disrupts receptor-G protein interaction. Next, the events occurring at the initial stages of TRH-mediated signal transduction were correlated to prolactin (PRL) secretion in GH3cells. Colchicine (1 μM) and taxol (1 μM) inhibited the basal PRL secretion by 38 and 44, respectively. In addition, colchicine (1 μM) and taxol (1 μM) significantly inhibited TRH-stimulated PRL secretion. TRH-stimulated PRL secretion in control, colchicine-, and taxol-treated cells was 13.9, 9.1, and 6 ng/mL, respectively. Furthermore, polymerized tubulin levels were decreased by colchicine and increased by taxol. These results suggest that perturbation of the steady state of tubulin-Gqinteraction may disrupt the initial events in TRH-mediated signal trans
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