Tubulin as a regulator of G-protein signaling.

Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses methods for examining this interaction.

Clathrin-mediated endocytosis of m3 muscarinic receptors. Roles for Gbetagamma and tubulin.

Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether Galpha(q) or Gbetagamma undergo such regulated translocation. Recent studies demonstrate that m3 muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates Galpha(q) and activates phospholipase Cbeta1. Interaction of tubulin-GDP with Gbetagamma at the offset of phospholipase Cbeta1 signaling appears involved in translocation of tubulin and Gbetagamma to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299-34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m3 muscarinic receptors or Galpha(q) involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of Galpha(q), Gbetagamma, tubulin, clathrin, and m3 receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, Galpha(q) and Gbetagamma. Fifteen minutes later receptors, Gbetagamma and tubulin, but not Galpha(q), internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m3 receptors with Gbetagamma, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that Gbetagamma subunits might organize the formation of a multiprotein complex linking m3 receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but beta-arrestin-independent endocytosis of m3 muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other beta-arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.

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.

Phosphatidylinositol 4,5-bisphosphate modifies tubulin participation in phospholipase Cbeta1 signaling.

Tubulin forms the microtubule and regulates certain G-protein-mediated signaling pathways. Both functions rely on the GTP-binding properties of tubulin. Signal transduction through Galpha(q)-regulated phospholipase Cbeta1 (PLCbeta1) is activated by tubulin through a direct transfer of GTP from tubulin to Galpha(q). However, at high tubulin concentrations, inhibition of PLCbeta1 is observed. This report demonstrates that tubulin inhibits PLCbeta1 by binding the PLCbeta1 substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Tubulin binding of PIP2 was specific, because PIP2 but not phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, or inositol 1,4,5-trisphosphate inhibited microtubule assembly. PIP2 did not affect GTP binding or GTP hydrolysis by tubulin. Muscarinic agonists promoted microtubule depolymerization and translocation of tubulin to the plasma membrane. PIP2 augmented this process in both Sf9 cells, containing a recombinant PLCbeta1 pathway, and SK-N-SH neuroblastoma cells. Colocalization of tubulin and PIP2 at the plasma membrane was demonstrated with confocal laser immunofluorescence microscopy. Although tubulin bound to both Galpha(q) and PLCbeta1, PIP2 facilitated the interaction between tubulin and PLCbeta1 but not that between tubulin and Galpha(q). However, PIP2 did augment formation of tubulin--Galpha(q)-PLCbeta1 complexes. Subsequent to potentiating PLCbeta1 activation, sustained agonist-independent membrane binding of tubulin at PIP2- and PLCbeta1-rich sites appeared to inhibit Galpha(q) coupling to PLCbeta1. Furthermore, colchicine increased membrane-associated tubulin and also inhibited PLCbeta1 activity in SK-N-SH cells. Thus, tubulin, depending on local membrane concentration, may serve as a positive or negative regulator of phosphoinositide hydrolysis. Rapid changes in membrane lipid composition or in the cytoskeleton might modify neuronal signaling through such a mechanism.