Effect of transient overexpression of Gqα on soluble and polymerized tubulin pools in GH3 and AtT-20 cells

Abstract

In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gqα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gqα-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gqα-specific staining was much more prominent in Gqα-transfected GH3 and AtT-20 cells (also transfected with Gqα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gqα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in Gqα-transfected GH1 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in Gqα-transfected GH3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by Gq directly, we examined the influence of purified Gq on tubulin polymerization. Gq (0.5 μM) inhibited polymerization of crude tubulin (present in GH3 cell cytosol) by 53%. In contrast to its effects on GH3 cell cytosol tubulin, Gq stimulated purified tubulin polymerization by 160%. These results suggest that Gq modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc.