Mark M Rasenick

Publications of Mark M Rasenick

• A molecular and structural mechanism for G protein-mediated microtubule destabilization.

  Authors: Rahul H Davé, Witchuda Saengsawang, Manu Lopus, Sonya Davé, Leslie Wilson, Mark M Rasenick

  The Journal of biological chemistry. 02/2011; 286(6):4319-28.

  The heterotrimeric, G protein-coupled receptor-associated G protein, Gα(s), binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activatedThe heterotrimeric, G protein-coupled receptor-associated G protein, Gα(s), binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of Gα(s) binds tubulin with a K(D) of 100 nm, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the α3-β5 region of Gα(s) is a functionally important motif in the Gα(s)-mediated microtubule destabilization. Indeed, peptides corresponding to that region of Gα(s) mimic Gα(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 Gα(s)/tubulin interface in which Gα(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 Gα(s) and the role of Gα(s) as a GTPase activator for tubulin. Given the ability of Gα(s) to translocate intracellularly in response to agonist activation, Gα(s) may play a role in hormone- or neurotransmitter-induced regulation of cellular morphology.

• Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors.

  Authors: Kumar U Kotlo, Mark M Rasenick, Robert S Danziger

  Molecular and cellular biochemistry. 12/2009;

  Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides andGuanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC's (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (alpha/beta) beta1 isoform of the beta subunit of NOGC (NOGCbeta1) was specifically focused. NOGCbeta1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCbeta1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCbeta1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 +/- 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 +/- 3.4 vs. 1.2 +/- 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCbeta1 abundance was reduced using specific siRNA to NOGCbeta1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCbeta1 (530.2 +/- 141.4 vs. 26.1 +/- 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCbeta1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 +/- 22 vs. 92 +/- 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCbeta1 and GC-A interact and that NOGCbeta1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.

• Chronic treatment with Escitalopram, but not R-citalopram translocates G{alpha}s from lipid raft domains and potentiates adenylyl cyclase: A 5-HT transporter independent action of this antidepressant compound.

  Authors: Lanqiu Zhang, Mark M. Rasenick

  The Journal of pharmacology and experimental therapeutics. 12/2009;

  Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galphas from lipid rafts to a non-raft fraction of the plasma membrane whereChronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galphas from lipid rafts to a non-raft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants and, in the case of serotonin uptake inhibitors, occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Galphas in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Galphas in lipid rafts, while there was no change in overall Galphas content. These effects were drug dose- and exposure time-dependent. While R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Galphas localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this appeared due to increased coupling between Galphas and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Galphas from lipid rafts and this effect appears to occur independently of 5-HT transporters. Our results suggest that, although antidepressants display distinct affinities for well-identified targets (e.g. monoamine transporters), several pre- and postsynaptic molecules are likely modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.

• Caveolin-1 and lipid microdomains regulate Gs trafficking and attenuate Gs/adenylyl cyclase signaling.

  Authors: John A Allen, Jiang Z Yu, Rahul H Dave, Anushree Bhatnagar, Bryan L Roth, Mark M Rasenick

  Molecular pharmacology. 09/2009;

  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 regulateLipid 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 beta-adrenergic receptor/Galphas 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 if caveolin-1 and lipid microdomains regulate Galphas trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either acute cholesterol chelation using methyl-beta-cyclodextrin or by stable knock-down of caveolin-1 and 2 by RNAi. In imaging studies examining Galphas-GFP during signaling, stimulation with the betaAR agonist isoproterenol resulted in internalization of Galphas-GFP; however, this trafficking was blocked by methyl-beta-cyclodextrin or by caveolin knock-down. Caveolin knock-down significantly decreased Galphas localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of Galphas to these membrane microdomains. Methyl-beta-cyclodextrin or caveolin knock-down significantly increased isoproterenol or TSH-stimulated cAMP accumulation. Furthermore, forskolin and aluminum tetrafluoride stimulated adenylyl cyclase activity was significantly increased by caveolin knock-down in cells or in brain membranes obtained from caveolin-1 knock-out mice, indicating caveolin attenuates signaling at the level of Galphas/adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on Galphas trafficking and signaling. It is suggested that lipid rafts/caveolae are sites which remove Galphas from membrane signaling cascades and caveolins might dampen globally Galphas/adenylyl cyclase/cAMP signaling.

• Cytosolic Galpha s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth.

  Authors: Jiang-Zhou Yu, Rahul H Dave, John A Allen, Tulika Sarma, Mark M Rasenick

  The Journal of biological chemistry. 03/2009;

  It is now evident that Galphas is trafficks into cytosol following GPCR activation and alpha subunits of some heterotrimeric G-proteins including Galphas bind to tubulin in vitro. Nevertheless, manyIt is now evident that Galphas is trafficks into cytosol following GPCR activation and alpha subunits of some heterotrimeric G-proteins including Galphas bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein alpha subunits remain unclear. In this study, several biochemical approaches demonstrated that activated-Galphas directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Galphas colocalized with microtubules. The activated, GTP-bound, Galphas mimicked tubulin in serving as a GTPase activator for beta-tubulin. As a result, activated-Galphas 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 Galphas 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-Galphas or activation of Galphas with cholera toxin in PKA-deficient PC12 cells, promoted neurite outgrowth in a cAMP independent manner. Thus, it is suggested that activated-Galphas 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.

• Heterotrimeric G-proteins interact directly with cytoskeletal components to modify microtubule-dependent cellular processes.

  Authors: Rahul H Dave, Witchuda Saengsawang, Jiang-Zhou Yu, Robert Donati, Mark M Rasenick

  Neuro-Signals. 02/2009; 17(1):100-8.

  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 messengerA 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 Galpha in membrane microdomains (such as lipid rafts) or the cytosol. Tubulin binds with nanomolar affinity to Gsalpha, Gialpha1 and Gqalpha (but not other Galpha subunits) as well as Gbeta(1)gamma(2) subunits. Galpha subunits destabilize microtubules by stimulating tubulin's GTPase, while Gbetagamma subunits promote microtubule stability. The same region on Gsalpha that binds adenylyl cyclase and Gbetagamma also interacts with tubulin, suggesting that cytoskeletal proteins are novel Galpha effectors. Additionally, intracellular Gialpha-GDP, in concert with other GTPase proteins and Gbetagamma, 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.

• Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins.

  Authors: Sukla Roychowdhury, Mark M Rasenick

  The FEBS journal. 09/2008;

  Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules andHeterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between G protein subunits (alpha and betagamma) and microtubules, and the molecular details emerging from these studies suggest that alpha and betagamma subunits of G proteins interact with tubulin/microtubules to regulate the assembly/dynamics of microtubules, providing a novel mechanism for hormone- or neurotransmitter-induced rapid remodeling of cytoskeleton, regulation of the mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation in which structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission.

• Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin.

  Authors: Brian T Layden, Witchuda Saengsawang, Robert J Donati, Shuo Yang, Debbie C Mulhearn, Michael E Johnson, Mark M Rasenick

  Biochimica et biophysica acta. 07/2008; 1783(6):964-73.

  A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block ofA number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.

• Postmortem brain tissue of depressed suicides reveals increased Gs alpha localization in lipid raft domains where it is less likely to activate adenylyl cyclase.

  Authors: Robert J Donati, Yogesh Dwivedi, Rosalinda C Roberts, Robert R Conley, Ghanshyam N Pandey, Mark M Rasenick

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 04/2008; 28(12):3042-50.

  Recent in vivo and in vitro studies have demonstrated that Gs alpha migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain inRecent in vivo and in vitro studies have demonstrated that Gs alpha migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain in response to chronic, but not acute, treatment with tricyclic or selective serotonin reuptake inhibitor antidepressants. This migration resulted in a more facile association with adenylyl cyclase. Our hypothesis is that Gs alpha may be ensconced, to a greater extent, in lipid rafts during depression, and that one action of chronic antidepressant treatment is to reverse this. In this postmortem study, we examined Gs alpha membrane localization in the cerebellum and prefrontal cortex of brains from nonpsychiatric control subjects and suicide cases with confirmed unipolar depression. Sequential TX-100 and TX-114 detergent extractions were performed on the brain tissue. In the cerebellum, the ratio of TX-100/TX-114-soluble Gs alpha is approximately 2:1 for control versus depressed suicides. Results with prefrontal cortex samples from each group demonstrate a similar trend. These data suggest that depression localizes Gs alpha to a membrane domain (lipid rafts) where it is less likely to couple to adenylyl cyclase and that antidepressants may upregulate Gs alpha signaling via disruption of membrane microenvironments. Raft localization of Gs alpha in human peripheral tissue may thus serve as a biomarker for depression and as a harbinger of antidepressant responsiveness.

• G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells.

  Authors: Valentina Montoya, Christina Gutierrez, Omar Najera, Denisse Leony, Armando Varela-Ramirez, Juliana Popova, Mark M Rasenick, Siddhartha Das, Sukla Roychowdhury

  Cell motility and the cytoskeleton. 01/2008; 64(12):936-50.

  The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interactsThe betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.

• Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts.

  Authors: Jun Sugama, Jiang-Zhou Yu, Mark M Rasenick, Norimichi Nakahata

  Cellular signalling. 12/2007; 19(11):2247-54.

  Mastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediatedMastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediated signaling, we previously examined the effect of mastoparan on G(q)-mediated signaling and demonstrated that mastoparan binds to gangliosides causing a decrease in Galpha(q/11) content in lipid rafts, and resulting in the inhibition of G(q)-mediated phosphoinositide hydrolysis (Sugama et al., Mol. Pharmacol., 68, 1466, 2005). In the present study, we examined the effect of mastoparan on beta-adrenoceptor-G(s) signaling in 1321N1 human astrocytoma cells. Mastoparan inhibited isoproterenol-induced elevation of cyclic AMP in a concentration-dependent manner. Although mastoparan is known to be an activator of G(i), pertussis toxin only slightly attenuated mastoparan-induced inhibition of cyclic AMP elevation, suggesting that a major part of the inhibition of cyclic AMP elevation induced by mastoparan is not mediated by Galpha(i). By contrast, mastoparan-induced inhibition of cyclic AMP elevation was clearly attenuated by preincubation of the cells with ganglioside mixtures. Moreover, mastoparan changed the localization of Galpha(s) in lipid rafts without disrupting the structure of lipid rafts. Fluorescent staining analysis showed that mastoparan released GFP-Galpha(s) from plasma membranes into the cytosol. These results suggest that the mastoparan-induced suppression of cyclic AMP elevation is mainly caused by changing the localization of Galpha(s) in lipid rafts into a compartment in the cellular interior where it is not available to activate adenylyl cyclase.

• Lipid raft microdomains and neurotransmitter signalling.

  Authors: John A Allen, Robyn A Halverson-Tamboli, Mark M Rasenick

  Nature reviews. Neuroscience. 03/2007; 8(2):128-40.

  Lipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membraneLipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membrane domains in which neurotransmitter signalling might occur through a clustering of receptors and components of receptor-activated signalling cascades. The localization of these proteins in lipid rafts, which is affected by the cytoskeleton, also influences the potency and efficacy of neurotransmitter receptors and transporters. The effect of lipid rafts on neurotransmitter signalling has also been implicated in neurological and psychiatric diseases.

• Tau associates with actin in differentiating PC12 cells.

  Authors: Jiang-Zhou Yu, Mark M Rasenick

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 08/2006; 20(9):1452-61.

  The microtubule-associated protein tau may be involved in cell morphogenesis and axonal maintenance. In addition to microtubules, tau has been shown to interact with actin in vitro. In the presentThe microtubule-associated protein tau may be involved in cell morphogenesis and axonal maintenance. In addition to microtubules, tau has been shown to interact with actin in vitro. In the present study interaction of tau and actin was investigated in PC12 cells. No interaction between tau and actin was observed without NGF treatment. Under NGF stimulation, tau distributed at ends of cellular extensions, where it associated with actin in a microtubule-independent manner. F-actin disruption revealed that relocalization and assembly of F-actin at the ends of cellular extensions were necessary for NGF-induced tau reorganization and association with actin. A truncated tau-GFP (tau(1-186)-GFP, N-terminal of tau) did not associate with actin. However, tau23(174-352)-GFP (carboxyl-terminal of Tau23) did associate with actin and the requirement for NGF was lost. Nevertheless, NGF boosted tau23(174-352)-GFP interaction with actin and promoted colocalization at the ends of cellular extensions. This suggests that the C-terminal of tau is required for associating with actin and the tau N-terminal may play a regulatory role in this process. A possible role for tau-actin interaction in neurite outgrowth is postulated.

• G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules.

  Authors: Sukla Roychowdhury, Liliana Martinez, Lucy Salgado, Siddhartha Das, Mark M Rasenick

  Biochemical and biophysical research communications. 03/2006; 340(2):441-8.

  Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that GHeterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.

• Chronic antidepressant treatment prevents accumulation of gsalpha in cholesterol-rich, cytoskeletal-associated, plasma membrane domains (lipid rafts).

  Authors: Robert J Donati, Mark M Rasenick

  Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 08/2005; 30(7):1238-45.

  Previous studies demonstrated that Gsalpha migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with thePrevious studies demonstrated that Gsalpha migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsalpha redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsalpha and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsalpha redistribution and the increased coupling between Gsalpha and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsalpha content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-beta-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsalpha localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsalpha out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsalpha as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsalpha and the cytoskeleton is involved in this process.

• Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells.

  Authors: John A Allen, Jiang Z Yu, Robert J Donati, Mark M Rasenick

  Molecular pharmacology. 06/2005; 67(5):1493-504.

  Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whetherUpon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of G alpha s, we developed a functional G alpha s-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing G alpha s-GFP were treated with 10 microM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, G alpha s-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing G alpha s-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that G alpha s does not traffic through common endocytic pathways. Furthermore, G alpha s-GFP did not colocalize with internalized beta2-adrenergic receptors, suggesting that G alpha s and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated G alpha s-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased G alpha s protein in Triton X-100 -insoluble membrane fractions, suggesting that G alpha s moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of G alpha s-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated G alpha s moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of G alpha s plays a specific role in G protein-coupled receptor-mediated signaling and could enable G alpha s to traffic into the cellular interior to regulate effectors at multiple cellular sites.

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

  Authors: Juliana S Popova, Mark M Rasenick

  The Journal of biological chemistry. 08/2004; 279(29):30410-8.

  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 demonstrateReceptors 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.

• Tubulin as a regulator of G-protein signaling.

  Authors: Mark M Rasenick, Robert J Donati, Juliana S Popova, Jiang-Zhou Yu

  Methods in enzymology. 02/2004; 390:389-403.

  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 cytoskeletonTubulin 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.

• G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling.

  Authors: Juliana S Popova, Mark M Rasenick

  The Journal of biological chemistry. 10/2003; 278(36):34299-308.

  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 GAgonist 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.

• Heterotrimeric G-proteins associate with microtubules during differentiation in PC12 pheochromocytoma cells.

  Authors: Tulika Sarma, Tatyana Voyno-Yasenetskaya, Thomas J Hope, Mark M Rasenick

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 06/2003; 17(8):848-59.

  Tubulin modifies G-protein signaling and heterotrimeric G-proteins regulate microtubule assembly. Here we report an interplay among G-protein-coupled receptor and receptor tyrosine kinase (such asTubulin modifies G-protein signaling and heterotrimeric G-proteins regulate microtubule assembly. Here we report an interplay among G-protein-coupled receptor and receptor tyrosine kinase (such as nerve growth factor-NGF) signaling systems in PC12 pheochromocytoma cells that resulted in a translocation of Galpha(s), Galpha(i1), and Galpha(o) from cell bodies to cellular processes where they appear to localize with tubulin-containing structures. This relocation appeared to depend on the integrity of microtubules, as it was blocked and reversed by nocodazole. Latrunculin, which promotes actin filament depolymerization, had no effect. Both deconvolution microscopy and immunoprecipitation showed a significant increase of Galpha association with microtubules that was coincident with the extension of "neurites." There were distinctions among the Galpha subtypes, with Galpha(s) showing the most profound NGF-induced colocalization with tubulin. Translocation of Galpha was blocked by agents that inhibit the MAP kinases required for neuronal differentiation, suggesting that G-protein relocation is triggered by the intracellular signals for differentiation. Consistent with this, Galpha in Neuro-2A cells, which spontaneously differentiate, showed a similar translocation coincident with differentiation. Thus, diverse signals that promote neuronal differentiation and changes in cell morphology may use specific G-proteins to evoke cytoskeletal rearrangement.