G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling.
Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.
Popova
Juliana S
JS
Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342, USA.
Rasenick
Mark M
MM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002217:Carbachol / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D005786:Gene Expression Regulation; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007527:Isoenzymes / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054799:Phospholipase C beta; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D015398:Signal Transduction; D013997:Time Factors; D015533:Transcriptional Activation; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D003432:Cross-Linking Reagents; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011838:Radiation-Sensitizing Agents; D014404:Tubulin; D002217:Carbachol; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D017239:Paclitaxel; D003078:Colchicine