MINIREVIEW Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins Sukla Roychowdhury 1 and Mark M. Rasenick 2 1 Neuroscience and Metabolic Disorder Unit, Border Biomedical Research Center and Department of Biological Sciences, University of Texas, El Paso, TX, USA 2 Department of Physiology and Biophysics, Psychiatry, University of Illinois, Chicago, IL, USA Microtubules constitute a crucial part of the cytoskele- ton and are involved in cell division and differentia- tion, cell motility, intracellular transport, and cell morphology [1,2]. These functions of microtubules are critically dependent upon the ability to polymerize and depolymerize. During mitosis, the interphase network of microtubules radiating throughout the cell changes into a bipolar spindle that mediates the accurate segregation of chromosomes. The half-life of micro- tubules changes from 5 to 10 min to 30 s to 1 min during this transition [3]. By contrast, the stability of microtubules increases significantly during differentia- tion [4]. The major component of microtubules is the heterodimeric protein, tubulin. Tubulin dimer binds Keywords cAMP; cytoskeleton; G protein-coupled receptor; G-protein; GTPase; microtubules; neurite outgrowth; RGS; synaptic plasticity; tubulin Correspondence M. M. Rasenick, Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott m ⁄ c 901, Chicago, IL 60612, USA Fax: +1 312 996 1414 Tel: +1 312 996 6641 E-mail: raz@uic.edu S. Roychowdhury, Department of Biological Sciences, University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968, USA Fax: +1 915 747 5808 Tel: +1 915 747 5943 E-mail: sukla@utep.edu (Received 15 April 2008, revised 18 July 2008, accepted 30 July 2008) doi:10.1111/j.1742-4658.2008.06614.x Heterotrimeric 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 (a and bc) and microtubules, and the molecu- lar details emerging from these studies suggest that a and bc 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. Abbreviations AGS3, activator of G protein signaling 3; GDI, guanine nucleotide dissociation inhibitors; Gia, alpha subunit of inhibitory G protein Gi; GoLoco motif, Gai ⁄ o-Loco interaction motif; GPCR, G protein-coupled receptors; GPR motif, G protein regulatory motif; Gbc, bc subunit of G protein; LGN, first identified as a Gai2-interacting protein and named LGN based on the presence of N-terminal Leu-Gly-Asn repeats; Loco, Drosophila Gia-interacting protein. 4654 FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 2 mol of GTP per mole of tubulin. Although both molecules of GTP are noncovalently bound, only one is exchangeable with free GTP (the E-site in b-tubulin). The presence of GTP enhances the polymerization pro- cess, and hydrolysis of GTP to GDP (most likely by an intrinsic tubulin GTPase) occurs subsequent to microtubule polymerization [5]. GTP hydrolysis by b-tubulin is a key element in determining the dynamic behavior of microtubules, and this hydrolysis creates a microtubule consisting largely of GDP–tubulin, but a small region of GTP-liganded tubulin, called a ‘GTP cap,’ remains at the end (Fig. 1). Loss of the cap results in the transition from growth to shortening (catastrophe), whereas re-acquisition of the GTP cap results in a transition from shortening to growth (res- cue) [6]. This characteristic dynamic behavior, termed ‘dynamic instability,’ allows a rapid remodeling of microtubules. An important consequence of dynamic instability is that it allows microtubules to search spe- cific target sites within the cell more effectively [7]. A large group of proteins known as microtubule-asso- ciated proteins are known to promote microtubule assembly and to stabilize microtubules both in vitro and in vivo (Fig. 1) [8–11]. Microtubule destabilization is achieved by a growing number of proteins, which include stathmin ⁄ Op18 (a small heat-stable protein that is abundant in many types of cancer cells), kata- nin, and some kinesin-related motor proteins [12,13]. These proteins have been shown to stimulate transi- tions from elongation to shortening of microtubules and are referred to as catastrophe-promoters (Fig. 1). Although much effort has been made in identifying and characterizing the cellular factors that regulate microtubule assembly and dynamics, the precise spatial and temporal control of the process is not clearly understood [14]. Heterotrimeric G proteins are comprised of a, b, and c subunits, with the former binding and hydrolyz- ing GTP. Activation of these G proteins follows ago- nist binding to a G protein-coupled receptor (GPCR) and binding of GTP to the Ga subunit. The activated Ga and Gbc modulate membrane-associated G protein effectors such as adenylyl cyclase, phospholipase, phos- phodiesterase or ion channels. GPCRs are activated by number of hormones, neurotransmitters and odorants and are coded for by a family or almost 1000 genes in humans. Similarly, several genes for G proteins exist and these code for 20 a subunits, 5 b subunits and 14 c subunits. G protein a subunits, which provide the primary determinant for ‘information flow’ from the activated GPCR are grouped into four families: Gs (for stimulatory), which activates adenylyl cyclase; Gi (inhibitory), which inhibits adenylyl cyclase (Gt, the photoreceptor G protein, transducins are also in this family); Gq, which activates phospholipase C; and G12 ⁄ 13, which is not discussed here. Note that there is a great deal of ‘flexibility’ in this system and G protein a and bc subunits are quite plastic in their activation of downstream effectors. Results obtained by us and others over nearly 30 years have revealed a complex between certain het- erotrimeric G protein alpha subunits (Gsa, Gi1a and Gqa) with a K d of 115–130 nm [15,16]. Tubulin has been shown to activate or inhibit adenylyl cyclase via the direct transfer of GTP to Gsa or Gia1 [17,18]. More relevant to this review, Gsa and Gia have been shown to activate tubulin GTPase and, in doing so, modulate microtubule dynamics [19]. This review focuses on our current understanding of G protein- regulated microtubule assembly and the cellular and physiological aspects of this regulation. Beyond transmembrane signaling: the interaction of G proteins with microtubules Although heterotrimeric G proteins are well known for their function in the downstream signaling of GPCRs, MAPs Kinesin-related Motor proteins Polymerization Depolymerization Stathmin/Op18 Nocodazole γ γ -tubulin Tubulin-GTP Tubulin-GDP Microtubules with GTP Cap Fig. 1. Polymerization ⁄ depolymerization of microtubules. Microtu- bules are polymerized from dimeric tubulin. GTP binding to tubulin is necessary for microtubule assembly to occur. GTP is hydrolyzed to GDP when tubulin is incorporated within the microtubule. In microtubules, GDP is bound to tubulin except at the plus (+) end where tubulin is still in the GTP-bound form, establishing the GTP cap. This cap allows microtubules to polymerize. When the cap is lost, microtubules begin to shrink. Microtubule-associated proteins (MAPs) are known to promote microtubule assembly and stabilize microtubules. The protein c-tubulin, a highly conserved centrosomal protein and member of the tubulin superfamily, plays a critical role in microtubule nucleation throughout the cell cycle. Stathmin ⁄ Op18, katanin, and some kinesin-related motor proteins are involved in microtubule depolymerization. These proteins have been shown to stimulate transitions from elongation to shortening of microtubules and are referred to as catastrophe-promoters. S. Roychowdhury and M. M. Rasenick G proteins and microtubule assembly FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 4655 evidence indicates that G proteins associate with sev- eral subcellular compartments, including microtubules, and participate in both cell division and differentiation [20–27]. For example, G protein b subunit antisense oligonucleotides have been shown to inhibit cell prolif- eration and to disorganize the mitotic spindle in mam- malian cells [21]. A nontraditional G protein signaling pathway has been shown to be involved in regulating the mitotic spindle for centrosome ⁄ chromosome move- ments in cell division in Caenorhabditis elegans, Droso- phila, and mammals. Components of this pathway include several proteins, including the Gi class of G proteins, GoLoco domain-containing proteins i.e. mammalian N-terminal Leu–Gly–Asn repeats (LGN) and activator of G protein signaling 3 (AGS3), regula- tors of G protein signaling (RGS), nuclear mitotic apparatus protein (NUMA), and resistors to inhibitors (RIC) of cholinesterase 8A [28–36]. Whereas Gia was shown to regulate microtubule pulling forces for chro- mosome movements, Gbc was found to be involved in spindle position and orientation. Several GPCRs, known to trigger neurite outgrowth have been identi- fied. These receptors are coupled to Gi ⁄ o, G12 ⁄ 13 or Gs families of G proteins [37–41]. However, the down- stream signaling involved in GPCR-triggered neurite outgrowth is not fully understood. A significant increase in Ga (Gi, Go and Gs) association with microtubules has been observed during nerve growth factor-induced differentiation of PC12 cells that was coincident with the extension of ‘neurites’ [26]. Similar results have been observed in Neuro-2A cells, which spontaneously differentiate. These results indicate that signals that promote cell division and differentiation may use specific G proteins for microtubule rearrange- ments. Thus, G proteins appear to provide a link between hormones or neurotransmitters and cell divi- sion, differentiation, and microtubules. Clustering of G proteins in lipid rafts and internalization of activated G alpha and Gbc Although G proteins are usually confined to the plasma membrane, translocation of activated Gsa and Gbc from the membrane to the cytosol has been observed [42–47]. It is possible that these proteins par- ticipate in localized regulation of the cytoskeleton, but the mechanism that governs the cellular destinations of G protein is not clearly understood. Lipid rafts (plasma membrane microdomains rich in cholesterol and sphingolipids) are thought to play key roles in G protein trafficking to subcellular compartments [48]. Many G proteins have been reported to localize to lipid rafts and undergo signal-dependent trafficking in to and out of lipid rafts. We have shown that Gsa is endocytosed by a lipid raft-mediated mechanism [49,50]. Unlike Ga,Gbc, was shown to internalize to cytosol with clathrin-coated vesicles [47]. Regulation of microtubule assembly by a and bc subunits of G proteins Studies conducted over the past few years have demon- strated that a and bc subunits of heterotrimeric G proteins modulate microtubule assembly in vitro [19,51,52]. Ga (Gi1a,Gsa,Goa) inhibits microtubule assembly and increases microtubule disassembly by activating the intrinsic GTPase of tubulin [19]. Thus, Ga may act as a GTPase-activating protein for tubulin and may increase the dynamic behavior of microtu- bules by removing the GTP cap [19], which confers stability on microtubules. The retinal G protein trans- ducin (Gta), which does not bind to tubulin [15], did not inhibit microtubule assembly or activate GTPase activity of tubulin [19]. In contrast to Ga,Gbc promotes microtubule assembly in vitro [51]. Specificity among bc species exists because b1c2 stimulates microtubule assembly and b1c1 is without effect. The prenylation state of G protein c subunits is likely to be relevant for this distinction (Gc1 is farnesylated, whereas Gc2 is gera- nylgeranylated). A mutant b1c2, b1c2 (C68S), which does not undergo prenylation and subsequent C-term- inal processing on the c subunit, does not stimulate the formation of microtubules [51]. Consistent with these observations, it has been suggested that lipid modification of G protein subunits (Ga and Gc) not only contributes to membrane association, but is also important for productive interactions between a with bc subunits, as well as the interactions of a and bc subunits with effector and receptor molecules [53,54]. For example, lipid modifications are critical for the interactions of a and bc subunits with effectors such as adenylyl cyclase, phospholipase C, and phosphati- dylinositol 3-kinase, as well as with receptors [55]. Our results suggested that the functional interactions of G protein subunits with tubulin⁄ microtubules require a similar structural specificity of G protein sub- units to those that determine their interactions with other signaling partners. Because G protein activation and subsequent dissociation of a and bc subunits is necessary for G proteins to participate in signaling processes, we reconstituted Gabc heterotrimer from myristoylated-Ga and prenylated-Gbc and found that the heterotrimer blocks the Gi1a activation of tubulin GTPase and inhibits the ability of Gb1c2 to promote G proteins and microtubule assembly S. Roychowdhury and M. M. Rasenick 4656 FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS in vitro microtubule assembly [52]. Nonetheless, G pro- tein heterotrimers bind to tubulin [56], suggesting that another site on Gbc (apart from the region binding to effector interaction domains on Ga) binds tubulin when the heterotrimer is intact. Thus, it appears that G protein activation and dissociation of a and bc sub- units is required for functional coupling between Ga ⁄ Gbc and tubulin ⁄ microtubules, as outlined in Fig. 2. In this model, Ga activates tubulin GTPase and destroys the GTP cap at microtubule ends, caus- ing an incease in microtubule dynamics. Thus, Ga is a GTPase activating protein for tubulin. In a sense, Ga is mimicking tubulin in the activation of the intrinsic tubulin GTPase. Because the predicted domain for interaction between Ga and tubulin is the interface where Ga interacts with effector [57,58], Ga ⁄ tubulin complexes preclude Gbc binding to Ga. It is likely that Ga and Gbc will interact with different populations of tubulin ⁄ microtubules to reorganize microtubule net- works in cells. Using the anti-mitotic agent nocodazole, we have shown that the assembly ⁄ disassembly of microtubules alters the tubulin–Gbc interaction in cultured PC12 and NIH3T3 cells [59]. Although microtubule depoly- merization by nocodazole inhibited the interactions between tubulin and Gbc , this inhibition was reversed when microtubule assembly was restored by the removal of nocodazole. The result suggests that Gbc might be involved in promoting microtubule assembly and ⁄ or stabilization of microtubules in vivo as demon- strated in vitro. This is further supported by the fact that Gbc was preferentially bound to microtubules and treatment with nocodazole (short-term incuba- tion), which suggested that the dissociation of Gbc from microtubules is an early step in the depolymeriza- tion process. Unlike Gbc, however, the interaction between tubulin and the a subunit of the Gs protein (Gsa) was not inhibited by nocodazole, which indicates differential interactions of the a and bc subunits of G proteins with tubulin ⁄ microtubules [59]. The anti- microtubule drugs nocodazole and colchicine are known to inhibit microtubule assembly by inhibiting the addition of tubulin dimers to microtubules [60,61]. The possibility that the anti-microtubule agent nocoda- zole exerts its effect by disrupting microtubule stabili- zation by Gbc may provide new understanding of the mechanism of action of the anti-mitotic ⁄ anti-cancer drugs and allow for the development of new drugs that might be more effective in the treatment of cancer. c-Tubulin–Gbc interactions and microtubule nucleation In addition to its binding of ab-tubulin, Gbc also interacts with c-tubulin in PC12 cells. However, unlike ab-tubulin, the interaction between c-tubulin and Gbc was not inhibited by nocodazole, suggesting that the interaction between Gbc and c-tubulin is not depen- dent upon microtubules. c-Tubulin is an integral cen- trosome protein, and its role in microtubule nucleation is well documented [62–64]. We found that Gbc was co-localized with ab- and c-tubulin in the centrosomes of PC12 cells [59]. The localization of Gbc in centro- somes and its association with c-tubulin suggest that Gbc might be involved in microtubule nucleation in association with c-tubulin (Fig. 2). This idea is sup- ported by in vitro observations, suggesting that Gbc promotes microtubule assembly under conditions where spontaneous nucleation does not occur [51]. Gα αβγ GPCRAgonist βγ G α No Effect G βγ Effectors Activation of GTPase of tubulin by G α , and inhibition of Loss of GTP cap on MT end by G α promo tes catastroph y di i Promotion of MT assembly by G βγ GTP-Tubulin MT assembly and increase in MT dynamics. MT with GTP cap G βγ GDP- Tubulin γ-Tub Fig. 2. Model for the regulation of microtubule (MT) assembly by a and bc subunits of G proteins. Based on in vitro results using puri- fied tubulin and G protein subunits (Ga,Gbc) [19,51,52], the follow- ing model is proposed. In this model, Ga inhibits microtubule assembly and promotes microtubule disassembly by interacting, in the fashion of a GTPase activating protein, with tubulin–GTP or the GTP cap of growing microtubules and initiating GTP hydrolysis of tubulin. Unlike the classical G protein cycle in which Ga in the GTP- bound form interacts with ‘effector’ molecules, this model shows that Ga interacts with tubulin ⁄ microtubules and this could be regu- lated by effector molecules or GAPs. Gbc, by contrast, promotes microtubule assembly. In the heterotrimer form, the primary inter- acting facets of Ga and Gbc are occluded. The Gabc heterotrimer can be activated either by agonist-mediated or agonist-independent pathways. Upon activation, Ga dissociates from Gbc subunits. Both subunits then interact with tubulin ⁄ microtubules and modulate assembly ⁄ dynamics. S. Roychowdhury and M. M. Rasenick G proteins and microtubule assembly FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 4657 Because it appears that microtubule nucleation by c-tubulin is mediated by the c-tubulin ring complex, the possibility exists that Gbc is a component of this complex [65,66]. It was previously shown that centro- some-associated c-tubulin is in a dynamic exchange with the cytoplasmic pool and that the c-tubulin con- tent of the centrosome increases suddenly, at least threefold, at the onset of mitosis [67]. In addition, the proportion of tubulin in microtubules increases drama- tically as the cell enters mitosis. However, the mechan- ism by which the translocation of c-tubulin and the subsequent activation of centrosomes occur is largely unknown. Microtubules do not appear to be involved in this dynamic exchange process [67]. We found that, in addition to c-tubulin, Gbc immunoreactivity also increased significantly in duplicated chromosomes at the onset of mitosis [59]. It can be speculated that Gbc may allow translocation of c-tubulin to centrosomes. The c-tubulin–Gbc complex might then induce robust microtubule nucleation at the centrosome and forma- tion of mitotic spindle. Cellular and physiological aspects of G protein–microtubule interactions Based on the above discussion, it can be speculated that G proteins may serve as a physiological regulator for microtubule assembly and dynamics. It is conceiva- ble that the interactions of Ga and Gbc with micro- tubules may modulate their dynamic behavior in cells. The results also suggest that GPCRs may affect regula- tion of microtubule assembly and dynamics in vivo by mobilizing G protein subunits to bind to microtubules. Certainly, in the case of Gsa there is clear evidence of agonist-induced translocation to the cytosol [45,49,68]. A number of proteins, in addition to GPCRs have been shown to influence the G protein activation cycle [69–72]. These proteins are identified as receptor-inde- pendent activators of G-protein signaling (AGS), and mediate a diverse range of signals within the cell, including cell division, neuronal differentiation and ⁄ or synaptic plasticity [71,72]. Three groups of AGS pro- teins have been defined based on their mechanism of action. Group I AGS protein (AGS1) is similar to that of a GPCR in terms of its ability to function as a gua- nine-nucleotide exchange factor. Group II and group III AGS proteins (AGS2-10) appear to regulate hetero- trimeric G protein signaling by a mechanism indepen- dent of nucleotide exchange. In contrast to group I and II AGS proteins, each member of the group III AGS proteins (AGS2, AGS7-10) binds to Gbc but not Ga. Group II AGS proteins (AGS3 ⁄ LGN) have been studied extensively. These proteins generally contain two types of repeats: tetratricopeptide repeats at the N-terminus that mediates protein–protein interactions, and Ga i ⁄ o -Loco (GoLoco or GPR) repeats at the C-terminus that mediate interactions with the Gi ⁄ o class of G proteins. Proteins containing G protein reg- ulatory (GPR) motifs have been identified in C. ele- gans (GPR1 ⁄ 2), Drosophila melanogaster (Pins), and mammalian cells (mammalian Pins or LGN; AGS3) [28]. These cytoplasmic signaling regulators have been described enzymatically as Gia-class guanine nucleo- tide dissociation inhibitors (GDI) that bind to the GDP bound form of Gia and inhibit the exchange of GDP-bound for GTP-bound Ga [73–75]. These signal- ing partners of G proteins might also be involved in the regulation of microtubule assembly by Gia or Gbc (Fig. 3). This is further supported by the fact that Ga in the GDP-bound form interacts with tubulin–GTP to promote the GTPase activity of tubulin and subsequent regulation of microtubule assembly [19]. Thus, the modulation of microtubule assembly by G proteins may require activation of G proteins by either recep- tor-dependent or receptor-independent pathways. Although molecules with GDI activity identified to date, only interact with Gi ⁄ o class of G proteins, it can be presumed that Gsa ⁄ Gqa-specific GDI molecules may be involved in regulating ⁄ modulating Gs or Gq ⁄ 11 family of G proteins, and thus may play roles in modulation of microtubule assembly by Gs or Gq ⁄ 11. Organization and function of mitotic spindle during cell division Transformation of an interphase network of micro- tubules into a bipolar spindle that mediates the accu- rate segregation of chromosomes is a central event during cell division. Microtubules in the spindle are organized in such a way that the minus ends are near the spindle poles and the plus ends extend toward the cell cortex or chromosomes [76]. Thus, the assembly ⁄ - disassembly of microtubules plays a key role in both the organization and function of the mitotic spindle. Recently, G protein subunits have been shown to be involved in regulating the mitotic spindle for centro- some ⁄ chromosome movements in cell division. Whereas Gia was shown to interact with GDI to regu- late microtubule pulling forces for chromosome move- ments, Gbc was found to be involved in spindle position and orientation. GoLoco domain-containing proteins (GDI) form complexes with Gia-GDP, which seems to create spindle oscillations by enhancing the pulling forces exerted on the mitotic spindle during mitosis [31]. Because it has been demonstrated pre- viously that Ga activates tubulin GTPase [19], it is G proteins and microtubule assembly S. Roychowdhury and M. M. Rasenick 4658 FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS possible that the direct interaction of microtubules with Ga- and LGN provides microtubule pulling forces through the destabilization of microtubules. Gbc, by contrast, may be involved in the orientation and positioning of the mitotic spindle through its abil- ity to interact with both membrane and centrosomes [29]. It can be speculated that Gbc is also involved in the formation of mitotic spindle by promoting micro- tubule assembly (in association with c-tubulin) in spin- dle poles. This is supported by the fact that at the onset of mitosis immunoreactivity of both c-tubulin and Gbc increased several fold in the duplicated cen- trosomes, thus increasing the capability of centrosomes to promote microtubule assembly [59]. Neuronal differentiation Microtubule assembly and dynamics is tightly coupled to neuronal differentiation, outgrowth, and plasticity. Several GPCR known to trigger neurite outgrowth have been identified [37–41]. However, the downstream signaling involved in GPCR-triggered neurite out- growth is not fully understood. The Go are the most abundant G proteins in neuronal growth cones [77]. Growth cones at the growing tips of developing neur- ites are highly specialized organelles that respond to a variety of extracellular signals to achieve neuronal gui- dance and target recognition [78]. These structures are associated with microtubules in their immature state, but microtubules retract from the tip of more mature growth cones. Some evidence suggests that Goa is directly involved in inducing neurite outgrowth upon activation [79]. By contrast, dendritic outgrowth pro- moted by the Gs-coupled GPR3, is cAMP-dependent [80]. Signaling through Gsa is also required for the growth and function of neuromuscular synapses in Drosophila [81]. Coordinated assembly of microtubules, in concert with actin filaments and neurofilaments, is required for growth cone motility and neurite out- growth [82–84] and microtubules in or near the growth cone are particularly dynamic [85]. Many functions of Go are thought to be mediated through the actions of a common pool of Gbc dimers. Based on the observed role of G protein subunits in microtubule assembly, it is reasonable to postulate that the dynamic interactions between Gi ⁄ o (both a and bc subunits) and microtubules, and the subsequent regula- tion of microtubule assembly may be critical for neuro- nal differentiation, outgrowth and plasticity. The G protein regulator AGS3, a Gia-class GDI, the expres- sion of which is restricted to neurons, might play a role in regulating the assembly ⁄ dynamics of microtubules in neurons by promoting the interactions between tubulin ⁄ microtubules and Gia-GDP. Association of Gbc with the actin cytoskeleton has also been reported [86]. More recent studies in cultured PC12 cells suggest that Gbc interacts with actin filaments in addition to microtubules and this interaction was not affected by depolymerization of microtubules (Najera & Roy- chowdhury, unpublished observations) and G proteins might serve to unite microtubule and actin-dependent processes to regulatory elements acting through GPCRs. A final caveat to the studies with Gi and Go is that, Agonis t G Membran e G Effector s GTP Cytoplasm GD P AG S AG S G (Group I a nd II) (Group III) GD P Microtubule dynamics G Fig. 3. G protein signaling in membrane and cytoplasm. Tradition- ally, G proteins function as a signal transducer in transmembrane signaling pathways that consist of three proteins: receptors, G pro- teins, and effectors. The receptors that participate in this pathway have seven transmembrane domains. G proteins consist of a het- erotrimeric structure composed of guanine nucleotide-binding alpha, plus beta and gamma subunits. Beta and gamma subunits form a tight association under nondenaturing conditions. Receptor activation allows GTP to bind to the a subunit of the heterotrimer. Subsequently, activated G a changes its association with Gbc in a manner that permits both subunits to participate in the regulation of intracellular effector molecules. Termination of the signal occurs when GTP bound to the a subunit is hydrolyzed by its intrinsic GTPase activity, which causes its functional dissociation from the effector and re-association with bc. A hypothetical framework for cytoplasmic G protein-signaling is shown. In this model, a and bc subunits of G proteins (only the Gsa are released from the membrane by agonist activation, but the Gia and Goa have a cyto- solic presence. All three, as well as Gq, evoke Gbc release into the cytosol) regulate microtubule assembly ⁄ dynamics (red arrows). By forming an inactive Gabc heterotrimer, this signaling pathway is ter- minated (purple arrows). In this model, AGS proteins will modulate the assembly ⁄ disassembly of microtubules by interacting with a and bc subunits of G proteins. Lipid modification of G protein sub- units, i.e. the myristoylation of Ga and prenylation of Gc, are expected to play key roles in microtubule regulation, similar to that observed with G protein signaling in membrane [53,54] (not shown in the model). Through this mechanism, GPCR might be involved in regulating the interplay of Gi1a,Gbc, AGS (or other Ga-interacting proteins) and tubulin ⁄ microtubules. S. Roychowdhury and M. M. Rasenick G proteins and microtubule assembly FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 4659 while both of these G proteins have a cytosolic presence and decorate microtubules [26], unlike Gs, they do not internalize in response to agonist. Nevertheless, GPCRs coupled to Gs, Gi, Go or Gq do evoke Gbc internaliza- tion [47,68,87]. This might suggest an interesting inter- play between Gs and Gi⁄ o (or Gq) in the regulation of microtubules and the modulation of cellular processes dependent on microtubule dynamics. Based on the current literature, we propose a com- prehensive model outlining the G protein-mediated sig- naling in membrane and cytoplasm as depicted in Fig. 3. Although the major membrane-associated com- ponents of G protein signaling are now well-defined, the phenomenon of cytosolic G protein signaling is only beginning to emerge. We speculate that in cyto- plasm a and bc subunits of G protein interact to regu- late microtubule assembly. It is also proposed that AGS proteins regulate microtubule assembly through their interaction with Ga or Gbc (Fig. 3). It is quite possible that lipid modification of G protein subunits plays key roles in microtubule regulation, similar to that observed with G protein signaling in membrane [52,53]. We speculate that G protein-coupled receptors will regulate the interplay of Ga Gbc, and AGS to modulate microtubule assembly. The interactions between receptor and non-receptor-mediated pathways in the regulation of G protein internalization are just beginning to be explored. It is becoming increasingly clear that a new pathway of cytosolic G protein signaling is emerging. We pro- pose that this pathway is involved in regulating micro- tubule dynamics. Hopefully, the next few years will bring new evidence that will elucidate the role of GPCR signaling in microtubule biology. These studies should help to establish the link between hormone or neurotransmitter action and modulation of cellular locomotion or cellular morphology. Acknowledgements Research in the authors’ laboratories described in this report was supported by MH 39595, AG015482 and DA020568 (MMR- U. Illinois Chicago), and 2G12RR08124 (University of Texas at El Paso). The authors like to thank Dr Siddhartha Das for critically reading the manuscript and thoughtful suggestions. Mr Traver Duarte and Mr Tavis Mendez are thanked for their help. References 1 Desai A & Mitchison TJ (1997) Microtubule polymeri- zation dynamics. Annu Rev Cell Dev Biol 13, 83–117. 2 Gelfand VI (1991) Microtubule dynamics: mechanism, regulation and function. Annu Rev Cell Biol 7, 93–116. 3 McNally FJ (1996) Modulation of microtubule dynamics during the cell cycle. Curr Opin Cell Biol 8, 23–29. 4 Bulinski JC & Gundersen GG (1991) Stabilization of post-translational modification of micotubules during cellular morphogenesis. Bio Essays 13, 285–293. 5 Carlier MF, Didry D, Simon C & Pantaloni D (1989) Mechanism of GTP hydrolysis in tubulin polymeriza- tion: characterization of the kinetic intermediate micro- tubule-GDP-Pi using phosphate analogues. Biochemistry 28, 1783–1791. 6 Mitchison T & Kirschner M (1984) Dynamic instability of microtubule growth. Nature 312, 237–242. 7 Gundersen GG, Gomes ER & Wen Y (2004) Cortical control of microtubule stability and polarization. Cur Opin Cell Biol 16, 106–112. 8 Murphy DB & Borisy GG (1975) Association of high- molecular weight proteins with microtubules and their role in microtubule assembly. Proc Natl Acad Sci USA 72, 2696–2700. 9 Margolis RL, Rauch CT & Job D (1986) Purification and assay of a 145-kDa protein (STOP145) with micro- tubule-stabilizing and motility behavior. Proc Natl Acad Sci USA 83, 639–643. 10 Gamblin TC, Nachmanoff K, Halpain S & Williams RC Jr (1996) Recombinant microtubule-associated pro- tein 2C reduces the dynamic instability of individual microtubules. Biochemistry 35, 12575–12586. 11 Bhat K & Setalu V (2007) Microtubule-associated pro- teins as targets in cancer chemotherapy. Clin Cancer Res 13, 2849–2854. 12 Belmont L & Mitchison TJ (1996) Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 84, 623–631. 13 Kline-Smith SL & Walczak CE (2002) The microtubule- destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells. Mol Biol Cell 13, 2718– 2731. 14 Gundersen GG & Cook TA (1999) Microtubules and signal transduction. Curr Opin Cell Biol 11, 81–94. 15 Wang N, Yan K & Rasenick MM (1990) Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1. J Biol Chem 265, 1239–1242. 16 Popova JS & Rasenick MM (2000) Muscarinic receptor activation promotes the membrane association of tubu- lin for the regulation of Gq-mediated phospholipase Cbeta(1) signaling. J Neurosci 15, 2774–2782. 17 Rasenick MM, Stein PJ & Bitensky MW (1981) The regulatory subunit of adenylate cyclase interacts with cytoskeletal components. Nature 294, 560–562. 18 Rasenick MM & Wang N (1988) Exchange of guanine nucleotides between tubulin and GTP-binding proteins that regulate adenylate cyclase: cytoskeletal G proteins and microtubule assembly S. Roychowdhury and M. M. Rasenick 4660 FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS modification of neuronal signal transduction. J Neuro- chem 51, 300–311. 19 Roychowdhury S, Panda D, Wilson L & Rasenick MM (1999) G protein alpha subunits activate tubulin GTPase and modulate microtubule polymerization dynamics. J Biol Chem 274, 13485–13490. 20 Wu HC & Lin CT (1994) Association of heterotrimeric GTP binding regulatory protein (Go) with mitosis. Lab Invest 71, 175–181. 21 Wu HC, Huang PH & Lin CT (2001) G protein beta2 subunit antisense oligonucleotides inhibit cell prolifera- tion and disorganize microtubule and mitotic spindle organization. J Cell Biochem 83, 136–146. 22 Lewis JM, Woolkalis MJ, Gerton GL, Smith RM, Jarett L & Manning DR (1991) Subcellular distribution of the alpha subunit(s) of Gi: visualization by immuno- fluorescent and immunogold labeling. Cell Regul 2, 1097–1113. 23 Ravindra R, Kunapuli SP, Forman LJ, Nagele RG, Foster KA & Patel SA (1996) Effect of transient over- expression of Gq alpha on soluble and polymerized tubulin pools in GH3 and AtT-20 cells. J Cell Biochem 61, 392–401. 24 Cote M, Payet MD & Gallo-Payet N (1997) Associa- tion of alpha S-subunit of the GS protein with micro- filaments and microtubules: implication during adrenocorticotropin stimulation in rat adrenalglomeru- losa cells. Endocrinology 138, 69–78. 25 Willard FS & Crouch MF (2000) Nuclear and cytoske- letal translocation and localization of heterotrimeric G-proteins. Immunol Cell Biol 78, 387–394. 26 Sarma T, Voyno-Yasenetskaya T, Hope TJ & Rasenick MM (2003) Heterotrimeric G-proteins associate with microtubules during differentiation in PC12 pheochro- mocytoma cells. FASEB J 17, 848–859. 27 Crouch MF & Simon L (1997) The G-protein G(i) reg- ulates mitosis but not DNA synthesis in growth factor- activated fibroblasts: a role for the nuclear translocation of G(i). FASEB J 11, 189–198. 28 Kimple RJ, Willard FS & Siderovski DP (2002) The GoLoco motif: heralding a new tango between G protein signaling and cell division. Mol Interven 2, 88–100. 29 Gotta M & Ahringer J (2001) Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos. Nat Cell Biol 3, 297–301. 30 Schaefer M, Petronczki M, Dorner D, Forte M & Knoblich J (2001) Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell 107, 183. 31 Fuse N, Hisata K, Katzen AL & Matsuzaki F (2003) Heterotrimeric G proteins regulate daughter cell size asymmetry in Drosophila neuroblast divisions. Curr Biol 13, 947–954. 32 Du Q & Macara IG (2004) Mammalian Pins is a con- formational switch that links NuMA to heterotrimeric G proteins. Cells 119, 503. 33 Sanada K & Tsai LH (2005) G protein betagamma sub- units and AGS3 control spindle orientation and asym- metric cell fate of cerebral cortical progenitors. Cell 122, 119–131. 34 Siegrist SE & Doe CQ (2005) Microtubule-induced Pins ⁄ G alpha I cortical polarity in Drosophila neuro- blasts. Cell 123, 1323–1335. 35 Bellaiche Y & Gotta M (2005) Heterotrimeric G pro- teins and regulation of size asymmetry during cell divi- sion. Curr Opin Cell Biol 17, 658–663. 36 Tall GG & Gilman AG (2005) Resistance to inhibitors of cholinerterase 8a catalyzes release of Gai-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA ⁄ LGN ⁄ Gai-GDP complexes. Proc Natl Acad Sci USA 102, 16584–16589. 37 Reinoso BS, Undie AS & Levitt P (1996) Dopamine receptors mediate differential morphological effects on cerebral cortical neurons in vitro. J Neurosci Res 43, 439–453. 38 Lotto B, Upton L, Price DJ & Gaspar P (1999) Seroto- nin receptor activation enhances neurite outgrowth of thalamic neurones in rodents. Neurosci Lett 269, 87–90. 39 He JC, Neves SR, Jordan JD & Iyengar R (2006) Role of the Go ⁄ i signaling network in the regulation of neur- ite outgrowth. Can J Physiol Pharmacol 84, 687–694. 40 Zhang W, Duan W, Cheung NS, Huang Z, Shao K & Li QT (2007) Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein- coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP genera- tion and neurite outgrowth in PC12 cells. J Neurochem 103, 1157–1167. 41 Kvachnina E, Liu G, Dityatev A, Renner U, Dumuis A, Richter DW, Dityateva G, Schachner M, Voyno- Yasenetskaya TA & Ponimaskin EG (2005) 5-HT7 receptor is coupled to G alpha subunits of heterotri- meric G12-protein to regulate gene transcription and neuronal morphology. J Neurosci 25, 7821–7830. 42 Rasenick MM, Wheeler GL, Bitensky MW, Kosack CM, Malina RL & Stein PJ (1984) Photoaffinity identi- fication of colchicine-solubilized regulatory subunit from rat brain adenylate cyclase. J Neurochem 43, 1447–1454. 43 Ransas LA, Svoboda JR, Jaspar JR & Insel PA (1989) Stimulation of beta-adrenergic receptors of S49 lym- phoma cells redistributes the alpha subunit of the stimu- latory G protein between cytosol and membranes. Proc Natl Acad Sci USA 86, 7900–7903. 44 Levis MJ & Bourne HR (1992) Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J Cell Biol 119, 1297–1307. S. Roychowdhury and M. M. Rasenick G proteins and microtubule assembly FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 4661 45 Yu JZ & Rasenick MM (2002) Real-time visualization of a fluorescent G(alpha)(s): dissociation of the acti- vated G protein from plasma membrane. Mol Pharma- col 61, 352–359. 46 Janetopoulos C, Jin T & Devreotes P (2001) Receptor- mediated activation of theterotrimeric G-proteins in liv- ing cells. Science 291, 2408–2411. 47 Popova JS & Rasenick MM (2004) Clathrin-mediated endocytosis of m3 muscarinic receptors. Roles for Gbetagamma and tubulin. J Biol Chem 279, 30410– 30418. 48 Allen JA, Halverson-Tamboli RA & Rasenick MM (2007) Lipid raft microdomains and neurotransmitter signalling. Nat Rev Neurosci 8, 128–140. 49 Allen JA, Yu JZ, Donati RJ & Rasenick MM (2005) Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells. Mol Pharmacol 67, 1493–1504. 50 Sugama J, Yu JZ, Rasenick MM & Nakahata N (2007) Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts. Cell Signal 19, 2247–2254. 51 Roychowdhury S & Rasenick MM (1997) G protein beta1gamma2 subunits promote microtubule assembly. J Biol Chem 272 , 31476–31581. 52 Roychowdhury S, Martinez L, Salgado L, Das S & Rasenick MM (2006) G protein activation is prerequi- site for functional coupling between Ga ⁄ Gbc and tubu- lin ⁄ microtubules. Biochem Biophys Res Commun 340, 441–448. 53 Mumby SM & Linder ME (1994) Myristoylation of G protein alpha subunits. Methods Enzymol 237, 254– 268. 54 Iniguez-LIuhi JA, Simon MI, Robinshaw JD & Gilman AG (1992) G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the signifi- cance of prenylation of gamma. J Biol Chem 267, 23409–23417. 55 Cabrera-Vera TM, Vanhauwe J, Thomas TO, Medkova M, Preininger A, Mazzoni MR & Hamm HE (2003) Insights into G proteinstructure, function, and regula- tion. Endrocrin Rev 24, 765–781. 56 Wang N & Rasenick MM (1991) Tubulin–G protein interactions involve microtubule polymerization domains. Biochemistry 30, 10957–10965. 57 Chen NF, Yu JZ, Skiba NP, Hamm HE & Rasenick MM (2003) A specific domain of Gialpha required for the transactivation of Gialpha by tubulin is implicated in the organization of cellular microtubules. J Biol Chem 278, 15285–15290. 58 Layden BT, Saengsawang W, Donati RJ, Yang S, Mul- hearn DC, Johnson ME & Rasenick MM (2008) Struc- tural model of a complex between the heterotrimeric G protein, Gsa, and tubulin. Biochim Biophys Acta 1783, 964–973. 59 Montoya V, Gutierrez C, Najera O, Leony D, Varela A, Popova J, Rasenick MM, Das S & Roychowdhury S (2007) G protein bc subunits interact with ab and c tubulin and play a role in microtubule assembly in PC12 cells. Cell Motil Cytoskel 64, 936–950. 60 DeBrabander MJ, Van de veire RML, Aerts FE, Borgers M & Janssen PAJ (1976) The effects of methyl [5-(2-thienylcarbonyl)-1H-benzimadazol-2-yl] carbamate (R17934; NSC 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells cultured in vitro. Cancer Res 36, 905–916. 61 De Brabander M, Geuens G, Nuydens R, Willebrords R & De MeyJ (1981) Microtubule assembly in living cells after release from nocodazole block: the effects of metabolic inhibitors, taxol and PH. Cell Biol Int Rep 5, 913–920. 62 Oakley BR (1992) Gamma-tubulin: the microtubule organizer? Trends Cell Biol 2, 1–5. 63 Joshi HC, Palacios MJ, McNamara L and Cleveland DW (1992) Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nuclea- tion. Nature 356, 80–83. 64 Job D, Valiron O & Oakley B (2003) Microtubule nucleation. Curr Opin Cell Biol 15, 111–117. 65 Moritz M, Braunfeld MB, Sedat JW, Alberts B & Agard DA (1995) Microtubule nucleation by gamma- tubulin-containing rings in the centrosome. Nature 378, 638–640. 66 Moritz M & Agard DA (2001) Gamma–tubulin com- plexes and microtubule nucleation. Curr Opin Struct Biol 11, 174–181. 67 Khodjakov A & Rieder CL (1999) The sudden recruit- ment of gamma-tubulin to the centrosome at the onset of mitosis, and its dynamic exchange throughout the cell cycle, does not require microtubules. J Cell Biol 146, 585–596. 68 Hynes TR, Mervine SM, Yost EA, Sabo JL & Berlot CH (2004) Live cell imaging of Gs and the beta2-adre- nergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor. J Biol Chem 279, 44101– 44112. 69 Bernard ML, Peterson YK, Chung P, Jourdan J & Lanier SM (2001) Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins. J Biol Chem 276, 1585–1593. 70 Blumer JB, Chandler J & Lanier SM (2002) Expression analysis and subcellular distribution of the two G-pro- tein regulators AGS3 and LGN indicate distinct func- tionality. J Biol Chem 277, 15897–15903. 71 Blumer JB, Cismowski MJ, Sato M & Lanier SM (2005) AGS proteins: receptor-independent activators of G-protein signaling. Trends Pharmacol Sci 26, 470–476. G proteins and microtubule assembly S. Roychowdhury and M. M. Rasenick 4662 FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 72 Blumer JB, Smrcka AV & Lanier SM (2007) Mechanis- tic pathways and biological roles for receptor-indepen- dent activators of G–perotein signaling. Pharmacol Ther 113, 488–506. 73 De Vries L, Fischer T, Tronchare H, Brothers GM, Strockbine B, Siderovski DP & Farquhar MG (2000) Activator of G protein signaling 3 is a guanine dissocia- tion inhibitor for Galphai subunits. Proc Natl Acad Sci USA 97, 14364–14369. 74 Peterson YK, Bernard ML, Ma H, Hazard S, Graber SG & Lanier SM (2000) Stabilization of the GDP- bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3. J Biol Chem 275, 33193–33196. 75 Willard FS, Kimple RJ & Siderovski DP (2004) Return of the GDI: the GoLoco motif in cell division. Annu Rev Biochem 73, 925–951. 76 Heidemann SR & McIntosh JR (1880) Visualization of the structural polarity of microtubules. Nature 286, 517–519. 77 Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ & Fishman MC (1990) G 0 is a major growth cone pro- tein subject to regulation by GAP-43. Nature 26, 836– 841. 78 Cheng N & Sahyoun N (1988) The growth cone cyto- skeleton. J Biol Chem 263, 3935–3942. 79 Igarashi M, Strittmatter S, Vartanian T & Fishman MC (1993) Mediation by G proteins of signals that cause collapse of growth cones. Science 259, 77–84. 80 Tanaka S, Ishii K, Kasai K, Yoon SO & Saeki Y (2007) Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. J Biol Chem 282, 10506–10515. 81 Wolfgang WJ, Clay C, Parker J, Delgado R, Labarca P, Kidokoro Y & Forte M (2004) Signaling through Gs alpha is required for the growth and function of neuro- muscular synapses in Drosophila. Dev Biol 268, 295– 311. 82 Smith S (1988) Neuronal cytomechanics: the actin based motility of growth cones. Science 242, 708–715. 83 Rodriguez OC, Schaefer AW, Mandato CA, Forscher P, Bement WM & Waterman-Storer CM (2004) Con- served microtubule–actin interactions in cell movement and morphogenesis. Curr Biol 14, 1194–1199. 84 Burnette DT, Schaefer AW, Ji L, Danuser G & Forscher P (2007) Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones. Nat Cell Biol 9, 1360–1369. 85 Suter DM, Schaefer AW & Forscher P (2004) Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering. Curr Biol 14, 1194–1199. 86 Carlson KE, Woolkalis MJ, Newhouse MG & Manning DR (1986) Fractionation of the beta subunit common to guanine nucleotide-binding regulatory proteins with the cytoskeleton. Mol Pharmacol 5, 463–468. 87 Saini DK, Kalyanaraman V, Chisari M & Gautam N (2007) A family of G protein betagamma subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation. J Biol Chem 282, 24099–24108. S. Roychowdhury and M. M. Rasenick G proteins and microtubule assembly FEBS Journal 275 (2008) 4654–4663 ª 2008 The Authors Journal compilation ª 2008 FEBS 4663 . inhibit the exchange of GDP-bound for GTP-bound Ga [73–75]. These signal- ing partners of G proteins might also be involved in the regulation of microtubule assembly by Gia or Gbc (Fig. 3). This is. I and II AGS proteins, each member of the group III AGS proteins (AGS2, AGS7-10) binds to Gbc but not Ga. Group II AGS proteins (AGS3 ⁄ LGN) have been studied extensively. These proteins generally. G Effector s GTP Cytoplasm GD P AG S AG S G (Group I a nd II) (Group III) GD P Microtubule dynamics G Fig. 3. G protein signaling in membrane and cytoplasm. Tradition- ally, G proteins