Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/ caveolae may regulate ␤-adrenergic receptor/G␣ s signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate G s trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-␤-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining G␣ s -GFP during signaling, stimulation with the ␤AR agonist isoproterenol resulted in internalization of G␣ s -GFP; however, this trafficking was blocked by methyl-␤-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased G␣ s localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of G␣ s to these membrane microdomains. Methyl-␤-cyclodextrin or caveolin knockdown significantly increased isoproterenol or thyrotropin-stimulated cAMP accumulation. Furthermore, forskolin-and aluminum tetrafluoride-stimulated adenylyl cyclase activity was significantly increased by caveolin knockdown in cells or in brain membranes obtained from caveolin-1 knockout mice, indicating that caveolin attenuates signaling at the level of G␣ s / adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on G␣ s trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove G␣ s from membrane signaling cascades and caveolins might dampen globally G␣ s / adenylyl cyclase/cAMP signaling.Lipid rafts and caveolae are specialized membrane microdomains defined by their cholesterol-and sphingomyelin-rich nature, enrichment in glycosyl-phosphatidylinositolanchored proteins, cytoskeletal association, and their resistance to detergent extraction (Brown, 2006). Lipid rafts and caveolae selectively partition and organize proteins and lipids in membranes, and they have been implicated in the regulation of a variety of cellular functions. These include exo-and endocytosis, membrane scaffolding, control of cholesterol homeostasis, and transmembrane signal transduction. A growing body of evidence indicates lipid rafts/caveolae regulate many G protein-coupled receptor (GPCR) signaling cascades by differentially partitioning GPCRs, heterotrimeric G proteins, and their various effectors in membrane microdomains (for reviews, see Allen et al., 2007;Patel et al., 2008). In addition to acting as organizing centers for signaling molecules, both lipid rafts and caveolae/caveolins can facilitate clathrin-independent endocytosis (Le Roy and Wrana, 2005; Rajendran and Si- Article, publication date, and citation information can be found at

It is now evident that G␣ s traffics into cytosol following G protein-coupled receptor activation, and ␣ subunits of some heterotrimeric G-proteins, including G␣ s bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein ␣ subunits remain unclear. In this study, several biochemical approaches demonstrated that activated G␣ s directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated G␣ s colocalized with microtubules. The activated, GTP-bound, G␣ s mimicked tubulin in serving as a GTPase activator for ␤-tubulin. As a result, activated G␣ s made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of G␣ s activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated G␣ s or activation of G␣ s with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated G␣ s acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.

A large percentage of current drugs target G-protein-coupled receptors, which couple to well-known signaling pathways involving cAMP or calcium. G-proteins themselves may subserve a second messenger function. Here, we review the role of tubulin and microtubules in directly mediating effects of heterotrimeric G-proteins on neuronal outgrowth, shape and differentiation. G-protein-tubulin interactions appear to be regulated by neurotransmitter activity, and, in turn, regulate the location of Gα in membrane microdomains (such as lipid rafts) or the cytosol. Tubulin binds with nanomolar affinity to Gsα, Giα1 and Gqα (but not other Gα subunits) as well as Gβ₁γ₂ subunits. Gα subunits destabilize microtubules by stimulating tubulin’s GTPase, while Gβγ subunits promote microtubule stability. The same region on Gsα that binds adenylyl cyclase and Gβγ also interacts with tubulin, suggesting that cytoskeletal proteins are novel Gα effectors. Additionally, intracellular Giα-GDP, in concert with other GTPase proteins and Gβγ, regulates the position of the mitotic spindle in mitosis. Thus, G-protein activation modulates cell growth and differentiation by directly altering microtubule stability. Further studies are needed to fully establish a structural mechanism of this interaction and its role in synaptic plasticity.

The heterotrimeric, G protein-coupled receptor-associated G protein, G␣ s , binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of G␣ s binds tubulin with a K D of 100 nM, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the ␣3-␤5 region of G␣ s is a functionally important motif in the G␣ s -mediated microtubule destabilization. Indeed, peptides corresponding to that region of G␣ s mimic G␣ s protein in activating tubulin GTPase and increase microtubule dynamic instability. We have identified specific mutations in peptides or proteins that interfere with this process. The data allow for a model of the G␣ s /tubulin interface in which G␣ s binds to the microtubule plus-end and activates the intrinsic tubulin GTPase. This model illuminates both the role of tubulin as an "effector" (e.g. adenylyl cyclase) for G␣ s and the role of G␣ s as a GTPase activator for tubulin. Given the ability of G␣ s to translocate intracellularly in response to agonist activation, G␣ s may play a role in hormone-or neurotransmitter-induced regulation of cellular morphology.