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

Abstract

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 (α and βγ) and microtubules, and the molecular details emerging from these studies suggest that α and βγ subunits of G proteins interact with tubulin/microtubules to regulate assembly/dynamics of microtubules, providing a novel mechanism for hormone or neurotransmitter induced rapid remodeling of cytoskeleton, regulation of mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation where structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission.

Introduction

Microtubules constitute a crucial part of the cytoskeleton and are involved in cell division and differentiation, 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 change into a bipolar spindle that mediates the accurate segregation of chromosomes. The half life of microtubules changes from 5–10 min to 30 s to 1 min during this transition [3]. On the other hand, the stability of microtubules increases significantly during differentiation [4]. The major component of microtubules is the heterodimeric protein, tubulin. Tubulin dimer binds 2 mol of GTP/mole of tubulin. Although both molecules of GTP are non-covalently bound, only one is exchangeable with free GTP (the E-site in β-tubulin). The presence of GTP enhances the polymerization process, and hydrolysis of GTP to GDP (most likely by an intrinsic tubulin GTPase) occurs subsequent to microtubule polymerization [5]. GTP hydrolysis by β-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 (Figure-1). The loss of the cap results in a transition from growth to shortening (catastrophe), whereas the re-acquisition of the GTP cap results in a transition from shortening to growing (rescue) [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 specific target sites within the cell more effectively [7]. A large group of proteins known as microtubule-associated proteins (MAPs) are known to promote microtubule assembly and to stabilize microtubules both in vitro and in vivo (Figure-1) [8–11]. ]. Microtubule destabilization is achieved by a growing number of proteins, which includes stathmin/Op18 (a small heat-stable protein that is abundant in many types of cancer cells), katanin, and some kinesin-related motor proteins [12, 13]. These proteins were shown to stimulate transitions from elongation to shortening of microtubules and are referred to as catastrophe-promoters (Figure-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 [reviewed in 14].

Figure 1. Polymerization/Depolymerization of microtubules.

Microtubules 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 microtubule. In microtubules, GDP is bound to tubulin except at the plus (+) end where tubulin is still in GTP bound form, establishing 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 γ-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 were shown to stimulate transitions from elongation to shortening of microtubules and are referred to as catastrophe-promoters

Heterotrimeric G proteins are comprised of α,β, and γ subunits, with the former binding and hydrolyzing GTP. Activation of these G proteins follows agonist binding to a G protein coupled receptor (GPCR) and binding of GTP to the Gα subunit. The activated Gα and Gβγ modulate membrane-associated G protein effectors such as adenylyl cyclase, phospholipase, and phosphodiesterase or ion channels. GPCRs are activated by number of hormones, neurotransmitters and odorants and are coded for by a family or nearly 1000 genes in humans. Similarly, several genes for G proteins exist and these code for twenty alpha subunits, 5 beta subunits and 14 gamma subunits. G protein alpha 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) inhibits adenylyl cyclase)—Gt, the photoreceptor G protein (the photreceptor G protein, transducins are also in this family); Gq, which activates phospholipase C; and G12/13, which will not be discussed in this review. Note that there is a great deal of “flexibility” in this system and G protein α and βγ subunits are quite plastic in their activation of downstream effectors.

Results obtained by us and others over nearly thirty years have revealed a complex between certain heterotrimeric G protein alpha subunits (Gsα, Gi1α and Gqα) with a Kd of 115–130 nM [15, 16]. Tubulin has been shown to activate or inhibit adenylyl cyclase via the direct transfer of GTP to Gsα or Giα1 [17, 18]. More relevant to this review, Gsα and Giα 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, evidence indicates that G proteins associate with several subcellular compartments, including microtubules, and participate in both cell division and differentiation [20–27]. For example, G protein β subunit antisense oligonucleotides have been shown to inhibit cell proliferation and to disorganize the mitotic spindle in mammalian cells [21]. A non-traditional G protein signaling pathway has been shown to be involved in regulating the mitotic spindle for centrosome/chromosome movements in cell division in C. elegans, Drosophila, and mammals. Components of this pathway include several proteins, including the Gi class of G proteins, GoLoco domain-containing proteins i.e. mammalian LGN (N-terminal Leu-Gly-Asn repeats) and AGS3 (Activator of G protein Signaling 3), RGS (regulators of G protein signaling), NuMA (nuclear mitotic apparatus protein), and resistors to inhibitors of cholinesterase (RIC) 8A [28–36]. While Giα was shown to regulate microtubule pulling forces for chromosome movements, Gβγ was found to be involved in spindle position and orientation. Several G protein coupled receptors (GPCR), known to trigger neurite outgrowth have been identified. These receptors are coupled to Gi/o, G12/13 or Gs families of G proteins [37–41]. However, the downstream signaling involved in GPCR-triggered neurite outgrowth is not fully understood. A significant increase in Gα (Gi, Go and Gs) association with microtubules was observed during NGF (Nerve Growth factor)-induced differentiation of PC12 cells that was coincident with the extension of “neurites” [26]. Similar results were 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 rearrangements. Thus, G proteins appear to provide a link between hormones or neurotransmitters and cell division, differentiation, and microtubules.

Clustering of G proteins in lipid rafts and internalization of activated G alpha and Gβγ

Although G proteins are usually confined to the plasma membrane, translocation of activated Gsα and Gβγ from the membrane to the cytosol has been observed [42–47] It is possible that these proteins participate 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 for review]. Many G proteins have been reported to localize to lipid rafts and undergo signal-dependent trafficking into and out of lipid rafts. We have shown that Gsα is endocytosed by a lipid raft-mediated mechanism [49, 50]. Unlike Gα, Gβγ, was shown to internalize to cytosol with clathrin-coated vesicles [47].

Regulation of microtubule assembly by α and βγ subunits of G proteins

Studies conducted over the past few years have demonstrated that α and βγ subunits of heterotrimeric G proteins modulate microtubule assembly in vitro [19, 51–52]. Gα (Gi1α, Gsα, Goα) inhibits microtubule assembly and increases microtubule disassembly by activating the intrinsic GTPase of tubulin [19]. Thus, Gα may act as a GAP (GTPase-activating protein) for tubulin and may increase dynamic behavior of microtubules by removing the GTP cap [19], which confers stability to microtubules. The retinal G protein transducin (Gtα), which does not bind to tubulin [15], did not inhibit microtubule assembly or activate GTPase activity of tubulin [19].

In contrast to Gα, Gβγ promotes microtubule assembly in vitro [51]. Specificity among βγ species exists because β1γ2 stimulates microtubule assembly and β1γ1 is without effect. The prenylation state of G protein γ subunits is likely to be relevant for this distinction (Gγ1 is farnesylated while Gγ2 is geranylgeranylated. A mutant β1γ2, β1γ2 (C68S), which does not undergo prenylation and subsequent carboxyterminal processing on the γ subunit, does not stimulate the formation of microtubules [51]. Consistent with these observations, it has been suggested that lipid modification of G protein subunits (Gα and Gγ) not only contributes to the membrane association but is also important for productive interactions between α with βγ subunits as well as the interactions of α and βγ subunits with effector and receptor molecules [53, 54]. For instance, lipid modifications are critical for the interactions of α and βγ subunits with effectors such as adenylyl cyclase (AC), phospholipase C (PLC), and PI3 kinase, as well as with receptors (see ref. 55 for review).

Our results suggested that the functional interactions of G protein subunits with tubulin/microtubules require a similar structural specificity of G protein subunits to those that determine their interactions with other signaling partners. Because G protein activation and subsequent dissociation of α and βγ subunits is necessary for G proteins to participate in signaling processes, we reconstituted Gαβγ heterotrimer from myristoylated-Gα and prenylated-Gβγ and found that the heterotrimer blocks Gi1α activation of tubulin GTPase and inhibits the ability of Gβ1γ2 to promote in vitro microtubule assembly [52]. Nonetheless, G protein heterotrimers bind to tubulin (56), suggesting that another site on Gβγ (apart from the region binding to effector interaction domains on Gα) binds tubulin when the heterotrimer is intact. Thus, it appears that G protein activation and dissociation of α and βγ subunits is required for functional coupling between Gα/Gβγ and tubulin/microtubules, as outlined in Figure 2. In this model, Gα activates tubulin GTPase and destroys the GTP-cap at microtubule ends. causing an incease in microtubule dynamics. Thus, Gα is a GAP, or GTPase Activating Protein for tubulin. In a sense, Gα is mimicking tubulin in the activation of the intrinsic tubulin GTPase. Since the predicted domain for interaction between Gα and tubulin is the interface where Gα interacts with effector [57, 58], Gα/tubulin complexes preclude Gβγ binding to Gα. It is likely that Gα and Gβγ will interact with different population of tubulin/MTs to reorganize microtubule networks in cells.

Figure 2. Model for the regulation of microtubule assembly by α and βγ subunits of G proteins.

Based on the in vitro results using purified tubulin and G protein subunits (Gα, Gβγ) (19, 51–52), the following model is proposed. In this model, Gα 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 Gα in GTP bound form interacts with “effector” molecules, this model shows that Gα interacts with tubulin/MTs and this could be regulated by effector molecules or GAPs. Gβγ, on the other hand promotes microtubule assembly. In the heterotrimer form, the primary interacting facets of Gα and Gβγ are occluded. The Gαβγ heterotrimer can be activated either by agonist-mediated or agonist-independent pathways. Upon activation, Gα dissociates from Gβγ subunits. Both subunits then interact with tubulin/microtubules and modulate assembly/dynamics.

Using the anti-mitotic agent nocodazole, we have shown that the assembly/disassembly of microtubules alters tubulin-Gβγ interaction in cultured PC12 and NIH3T3 cells [59]. While microtubule depolymerization by nocodazole inhibited the interactions between tubulin and Gβγ, this inhibition was reversed when microtubule assembly was restored by the removal of nocodazole. The result suggests that Gβγ might be involved in promoting microtubule assembly and/or stabilization of microtubules in vivo as demonstrated in vitro. This is further supported by the fact that Gβγ was preferentially bound to microtubules and treatment with nocodazole (short-term incubation), which suggested that the dissociation of Gβγ from microtubules is an early step in the depolymerization process. Unlike Gβγ, however, the interaction between tubulin and the α subunit of the Gs protein (Gsα ) was not inhibited by nocodazole, which indicates differential interactions of the α and βγ 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 nocodazole exerts its effect by disrupting microtubule stabilization by Gβγ 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.

γ-tubulin- Gβγ Interactions and Microtubule nucleation

In addition to its binding of αβ-tubulin, Gβγ also interacts with γ-tubulin in PC12 cells. However, unlike αβ-tubulin, the interaction between γ-tubulin and Gβγ was not inhibited by nocodazole, suggesting that the interaction between Gβγ and γ-tubulin is not dependent upon microtubules. γ—tubulin is an integral centrosome protein, and its role in microtubule nucleation is well documented [62–64]. We found that Gβγ was co-localized with αβ- and γ-tubulin in the centrosomes of PC12 cells [59]. The localization of Gβγ in centrosomes and its association withγ-tubulin suggest that Gβγ might be involved in microtubule nucleation in association with γ-tubulin (Figure 2). This idea is supported by in vitro observations, suggesting that Gβγ promotes microtubule assembly under conditions where spontaneous nucleation does not occur [51]. Because it appears that microtubule nucleation by γ-tubulin is mediated by γTuRC (γ-Tubulin Ring Complex), the possibility exists that Gβγ is a component of this complex [65, 66]. It was shown earlier that centrosome-associated γ-tubulin is in a dynamic exchange with the cytoplasmic pool and that the γ-tubulin content of the centrosome increases suddenly—at least three-fold—at the onset of mitosis [67]. In addition, the proportion of tubulin in microtubules dramatically increases as the cell enters mitosis. However, the mechanism by which the translocation of γ-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 γ-tubulin, Gβγ immuno-reactivity also increased significantly in duplicated chromosomes at the onset of mitosis [59]. It can be speculated that Gβγ may allow translocation of γ-tubulin to centrosomes. The γ-tubulin-Gβγ complex might then induce robust microtubule nucleation at the centrosome and formation 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 conceivable that the interactions of Gα and Gβγ with microtubules may modulate their dynamic behavior in cells. The results also suggest that GPCRs may effect regulation of microtubule assembly and dynamics in vivo by mobilizing G protein subunits to bind to microtubules. Certainly, in the case of Gsα 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-independent 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 [reviewed in 71, 72]. Three groups of AGS proteins 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 guanine-nucleotide exchange factor (GEF). Group II and group III AGS proteins (AGS2-10) appear to regulate heterotrimeriic G protein signaling by a mechanism independent 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 Gβγ but not Gα. Group II AGS proteins (AGS3/LGN) have been studied extensively. These proteins generally contain two types of repeats: tetratricopeptide repeats (TPR) at the amino-terminus that mediates protein-protein interactions, and Gα_i/o-Loco (GoLoco or GPR) repeats at the carboxy-terminus that mediate interactions with the Gi/o class of G proteins. Proteins containing GPR (G Protein Regulatory motif) motifs have been identified in C. elegans (GPR1/2), Drosophila melanogaster (Pins), and mammalian cells (mammalian Pins or LGN; AGS3) (please see 28 for review) These cytoplasmic signaling regulators have been described enzymatically as Giα-class GDIs (guanine nucleotide dissociation inhibitors) that bind to the GDP bound form of Giα and inhibit the exchange of GDP-bound for GTP-bound Gα [73–75]. These signaling partners of G proteins might also be involved in the regulation of microtubule assembly by Giα or Gβγ (Figure 2). This is further supported by the fact that Gα in 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 either by receptor-dependent or by receptor-independent pathways. Although molecules with GDI activity identified so far, only interacts with Gi/o class of G proteins, it can be presumed that Gsα/Gqα-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 inter-phase network of microtubules into a bipolar spindle that mediates the accurate 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 while 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 mitotic spindle. Recently, G protein subunits have been shown to be involved in regulating the mitotic spindle for centrosome/chromosome movements in cell division. While Giα was shown to interact with GDI to regulate microtubule pulling forces for chromosome movements, Gβγ was found to be involved in spindle position and orientation.. GoLoco domain-containing proteins (GDI) form complexes with Giα-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 previously that Gα activates tubulin GTPase [19], it is possible that the direct interaction of microtubules with Gα- and LGN provides microtubule pulling forces through destabilization of microtubules. Gβγ, on the other hand, may be involved in orientation and positioning of the mitotic spindle through its ability to interact with both membrane and centrosomes [29]. It can be speculated that Gβγ is also involved in the formation of mitotic spindle by promoting microtubule assembly (in association with γ-tubulin) in spindle poles. This is supported by the fact that at the onset of mitosis immuno-reactivity of both γ-tubulin and Gβγ increased several fold in the duplicated centrosomes, 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 G protein-coupled receptors (GPCR) known to trigger neurite outgrowth have been identified [37–41]. However, the downstream signaling involved in GPCR-triggered neurite outgrowth 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 neurites are highly specialized organelles that respond to a variety of extracellular signals to achieve neuronal guidance 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 Goα is directly involved in inducing neurite outgrowth upon activation [79]. On the other hand, dendritic outgrowth promoted by the Gs-coupled GPR3, is cAMP-dependent [80]. Signaling through Gsα 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 outgrowth [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 Gβγ 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 α and βγ subunits) and microtubules and the subsequent regulation of microtubule assembly may be critical for neuronal differentiation, outgrowth and plasticity. The G protein regulator AGS3, a Giα-class GDI, the expression 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 Giα-GDP. Association of Gβγ with the actin cytoskeleton has also been reported [86]. More recent studies in cultured PC12 cells suggest that Gβγ interacts with actin filaments in addition to microtubules and this interaction was not affected by depolymerization of microtubules (Najera and Roychowdhury, 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, while both of these G proteins have a cytosolic presence and decorate microtubules (26, 61), unlike Gs, they do not internalize in response to agonist. Nevertheless, GPCRs coupled to Gs, Gi, Go or Gq do evoke Gβγ internalization [47, 68, 87]. This might suggest an interesting interplay 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 comprehensive model outlining the G protein-mediated signaling in membrane and cytoplasm as depicted in Figure 3. While the major membrane-associated components of G protein signaling are now well-defined, the phenomenon of cytosolic G-protein signaling is only beginning to emerge. We speculate that in cytoplasm α and βγ subunits of G protein interact to regulate microtubule assembly. It is also proposed that AGS proteins regulate microtubule assembly through their interaction with Gα or Gβγ (Figure 3). It is quite possible that lipid modification of G protein subunits play 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 Gα Gβγ, 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.

Figure 3. G protein signaling in membrane and cytoplasm.

Traditionally, G proteins function as signal transducer in transmembrane signaling pathways that consists of three proteins: receptors, G proteins, and effectors. The receptors that participate in this pathway have seven transmembrane domains. G proteins consist of a heterotrimeric structure composed of guanine nucleotide binding alpha, plus beta and gamma subunits. Beta and gamma subunits form a tight association under non denaturing conditions. Receptor-activation allows GTP to bind to the α subunit of the heterotrimer. Subsequently, activated Gα changes its association with Gβγ in a manner that permits both subunits to participate in regulation of intracellular effector molecules. Termination of the signal occurs when GTP bound to the α subunit is hydrolyzed by its intrinsic GTPase activity, which causes its functional dissociation from the effector and re-association with βγ. A hypothetical framework for cytoplasmic G protein-signaling is shown. In this model, α and βγ subunits of G proteins (Only the Gsα are released from the membrane by agonist activation, but the Giα and Goα have a cytosolic presence. All three, as well as Gq, evoke Gβγ release into the cytosol.) regulate microtubule assembly/dynamics (red arrows). By forming an inactive Gαβγ heterotrimer, this signaling pathway is terminated (purple arrows). In this model, AGS proteins will modulate assembly/disassembly of microtubules by interacting with α and βγ subunits of G proteins. Lipid modification of G protein subunits i.e myristoylation of Gα and prenylation of Gγ 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, G protein-coupled receptors might be involved in regulating the interplay of Gi1α, Gβγ, AGS (or other Gα-interacting proteins) and tubulin/microtubules.

It is becoming increasingly clear that a new pathway of cytosolic G protein signaling is emerging. We propose that this pathway is involved in regulating microtubule 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.

Abbreviations

AGS3

Activator of G protein Signaling 3

GDI

Guanine nucleotide Dissociation Inhibitors

GPCR

G Protein Coupled Receptors

GPR motif

G Protein Regulatory motif

GTP

Guanosine-Tri-Phosphate

GDP

Guanosine-Di-Phosphate

Giα

alpha subunit of inhibitory G protein Gi

Gβγ

βγ subunit of G protein

GoLoco motif

Gαi/o-Loco interaction motif

Loco

Drosophila Giα-interacting protein

LGN

First identified as a Gαi2-interacting protein and named LGN based on the presence of N-terminal Leu-Gly-Asn repeats

MT

Microtubules

NGF

Nerve Growth Factor