Gβγ Mediates the Interplay between Tubulin Dimers and Microtubules in the Modulation of Gq Signaling

Abstract

Agonist stimulation causes tubulin association with the plasma membrane and activation of PLCβ1 through direct interaction with, and transactivation of, Gαq. Here we demonstrate that Gβγ interaction with tubulin down-regulates this signaling pathway. Purified Gβγ, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and Gαq transactivation by tubulin. Polymerization of microtubules elicited by Gβγ overrode tubulin translocation to the membrane in response to carbachol stimulation. Gβγ sequestration of tubulin reduced the inhibition of PLCβ1 observed at high tubulin concentration. Gβ1γ2 interacted preferentially with tubulin-GDP, whereas Gαq. was transactivated by tubulin-GTP. Prenylation of the α2 polypeptide was required for Gβγ/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of Gβγ/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells Gβγ 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 Gβ. Fifteen min post-carbachol addition, tubulin and Gβ colocalized in vesicle-like structures in the cytosol. Gβ/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 Gβγ/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLCβ1 signaling. Newly cytosolic Gβγ/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke Gα and Gβγ to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.