Effect of transient overexpression of Gq alpha on soluble and polymerized tubulin pools in GH3 and AtT-20 cells.
In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gq alpha cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gq alpha-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gq alpha-specific staining was much more prominent in Gq alpha-transfected GH3 and AtT-20 cells (also transfected with Gq alpha) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gq alpha. 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 Gq alpha-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 Gq alpha-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 microM) 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.
Ravindra
R
R
Department of Cell and Molecular Biology, University of Medicine and Dentistry of New Jersey-School of Osteopathic Medicine, Stratford 08084, USA.
Kunapuli
S P
SP
Forman
L J
LJ
Nagele
R G
RG
Foster
K A
KA
Patel
S A
SA
D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008954:Models, Biological; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000276:immunology / Q000378:metabolism*
D011108:Polymers; D014404:Tubulin; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D019204:GTP-Binding Proteins