Premium
Effect of transient overexpression of G qα on soluble and polymerized tubulin pools in GH 3 and AtT‐20 cells
Author(s) -
Ravindra R.,
Kunapuli S.P.,
Forman L.J.,
Nagele R.G.,
Foster K.A.,
Patel S.A.
Publication year - 1996
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19960601)61:3<392::aid-jcb6>3.0.co;2-o
Subject(s) - transfection , tubulin , microbiology and biotechnology , cytosol , microtubule , biology , cell culture , biochemistry , enzyme , genetics
In order to study G q ‐tubulin interaction in the cytosol, GH 3 and AtT‐20 cells (stably expressing TRH receptor) were transiently transfected with G qα cDNA. Forty‐eight hours after transfection, thyrotropin‐releasing hormone (TRH)‐stimulated prolactin (PRL) secretion by G qα ‐transfected GH 3 cells increased by 90% compared to mock‐transfected cells. In addition, using immunocytochemistry it was observed that G qα ‐specific staining was much more prominent in G qα ‐transfected GH 3 and AtT‐20 cells (also transfected with G qα ) compared to mock‐transfected cells. Thus, transfection resulted in successful overexpression of functional G qα . 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 qα ‐transfected GH 1 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 qα ‐transfected GH 3 and AtT‐20 cells. To determine whether these effects on tubulin were mediated by G q directly, we examined the influence of purified G q on tubulin polymerization. G q (0.5 μM) inhibited polymerization of crude tubulin (present in GH 3 cell cytosol) by 53%. In contrast to its effects on GH 3 cell cytosol tubulin, G q stimulated purified tubulin polymerization by 160%. These results suggest that G q 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.