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

R. Ravindra, S. P. Kunapuli, L. J. Forman, R. G. Nagele, K. A. Foster, S. A. Patel

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with G cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by G-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that G-specific staining was much more prominent in G-transfected GH3 and AtT-20 cells (also transfected with G) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional G. 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 G-transfected GH3 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 G-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.

Original languageEnglish (US)
Pages (from-to)392-401
Number of pages10
JournalJournal of Cellular Biochemistry
Volume61
Issue number3
DOIs
StatePublished - Jan 1 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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