G Proteins and Axon Growth

Seminars in NEUROSCIENCE 9, 209–219 (1998) Article No. SN970118

G Proteins and Axon Growth Kathleen L. Vancura and Daniel G. Jay1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138

This article highlights recent studies into the roles of the G proteins in two processes required for axon growth: growth cone motility and vesicular transport. Heterotrimeric G proteins are involved in growth cone motility, but their precise roles remain controversial. The small GTP-binding proteins are clearly established regulators of the actin cytoskeleton in fibroblasts, and their functions are just beginning to be explored in the growth cone. Members of the rab subfamily of small GTP-binding proteins have been shown to regulate vesicular transport in every cell type examined thus far, including neurons. r 1998 Academic Press

KEY WORDS: axon elongation; neuronal development; GTP-binding proteins; cytoskeleton; growth cone.

The formation of the nervous system presents unique problems of complex integration. Each neuron, by virtue of its location, neurochemistry, and morphology, is a distinct component that must be correctly interconnected to form functional neurocircuitry. Neurons are connected together by axons that must sometimes grow great distances throughout the organism. This growth requires specialized transport of material through the nascent axon as most of the proteins and membranes that comprise the axon are synthesized in the neuronal cell body. Axon growth throughout the embryo must be precisely guided so that neurons innervate their correct targets. The precise specification of axon growth requires exquisite control of intracellular processes by the neuron’s local environment, and this control is likely mediated by intracellular signal transduction. Recent studies have implicated a role for G proteins in this control, and this article summarizes this recent progress. In particular, different G proteins have been implicated in two processes required for axon growth: growth cone motility and vesicular transport. We will provide a brief overview of G proteins and describe experiments that implicate G proteins in these two processes. 1To whom correspondence should be addressed. E-mail: [email protected].

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A BRIEF OVERVIEW OF G PROTEINS Two major classes of GTP-binding proteins have been identified, heterotrimeric G proteins and small nucleotide-binding proteins (see Weng, et al., this issue). Heterotrimeric G proteins consist of three subunits—Ga (39–52 kDa), Gb (35–36 kDa), and Gg (7–10 kDa), although the Gb and Gg subunits are tightly associated and essentially act as a single subunit (reviewed in 1). Over 20 diverse Ga proteins are known to exist. They are classified into four major families—Gas,i,q, or 12/13. In mammals, five known b subunits and six g subunits are known. The b subunits are very similar to each other, exhibiting between 53 and 90% identity, whereas the g subunits exhibit greater diversity (reviewed in 1). The Ga subunit binds GTP when active, GDP when quiescent, and has an intrinsic GTPase activity that mediates the shift from the active to the quiescent state. A G protein complex consisting of Ga–GDP, Gb, and Gg is usually associated with an inactive transmembrane receptor. Ligand binding activates the receptor, inducing the Ga subunit’s exchange of GDP for GTP. The Gb–Gg heterodimer and the receptor then dissociate from Ga–GTP, allowing the subunits to interact with downstream effectors. These interactions amplify the initial, extracellular signal until Ga’s intrinsic GTPase hydrolysis activity is triggered, sometimes by

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a GTPase-activating protein. The bound GTP is hydrolyzed, and Ga–GDP is released from the effector. The Gb–Gg heterodimer now associates with Ga–GDP and forms the heterotrimeric complex that can interact with an activated receptor (2). The Ga subunits have been implicated in growth cone motility and neurite outgrowth. The other class of G proteins, small GTP-binding proteins, includes members of the ras-related superfamily. This family, in turn, can be subdivided into five subfamilies—ras, rho, rab, arf, and ran (3). Similar to heterotrimeric G proteins, the binding of GTP to one of these molecules serves to activate the protein and allows it to interact with downstream effectors. The hydrolysis of GTP serves to switch the protein ‘‘off.’’ This cycle is often facilitated by GTPase-activating proteins (GAPs, which stimulate GTPase activity), guanine nucleotide exchange proteins (GEPs, which aid the transfer of GDP for GTP), and guanine nucleotide dissociation inhibitors (GDIs, which prevent dissociation of GDP) (4).

Three of the five subfamilies, ras, rho, and rab, have been implicated in axon outgrowth thus far. Ras and rho family members help couple signal transduction at the plasma membrane to motile, cellular responses such as actin polymerization and lamellipodial ruffling. Members of the rab family are involved in vesicular transport (3), which is required to support axon elongation. Figure 1 summarizes some of the possible neuronal functions of the G proteins discussed below.

AXON GUIDANCE AND GROWTH CONE MOTILITY Axon growth is guided by the neuronal growth cone, a highly motile structure extending from the distal end of the axon (5). The growth cone moves in response to external cues with filopodia, finger-like processes extending from the growth cone body (6, 7)

FIG. 1. Proposed roles for G proteins in axon growth. Both heterotrimeric G proteins and small GTP-binding proteins have been implicated in the regulation of axon growth. Although the participation of a heterotrimeric G protein has been indicated, the role of the best candidate Gao remains unclear. Work in both neuronal and nonneuronal systems has suggested the involvement of members of the ras-related superfamily in actin-based motility and neurite outgrowth. Specifically, rho, rac, and cdc42 may participate in growth cone motility. In fibroblasts, activation of cdc42 leads to the formation of filopodia and to the stimulation of rac. Ras can also activate rac, which in turn gives rise to the formation of lamellipodia and the activation of rho. Active rho then causes neurite retraction. Various rab family members (namely, rab 2, 3a, 5a, and 8) participate in different steps of vesicular transport or endocytosis. Activation of rab2 results in increased spreading, adhesion, and neurite outgrowth by a mechanism that remains unclear. Although rab3a participates in synaptic vesicle fusion in mature neurons, the protein’s role in endocytosis in immature neurons is equivocal. The roles of rab5a and rab8 are clearer. Rab5a is implicated as a regulator of endocytosis, whereas rab8 is a key regulator of anterograde vesicle traffic in differentiating neurons.

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and with sheet-like extensions termed lamellipodia (8, 9; reviewed in 10). It is thought that the cues elicit signals that are integrated and then translated into a sequence of events resulting in changes in shape and motility of the growth cone (11–18). Growth cone motility is mediated by changes in the neuron’s underlying cytoskeleton, which is composed mainly of actin and tubulin. Actin microfilaments are the dominant cytoskeletal components of both lamellipodia and filopodia (19) and form a meshwork in the leading edge of the growth cone (reviewed in 7). Highly stabilized microtubules extend from the axon to the growth cone, whereas more dynamic microtubules spread out and cross the central region of the growth cone (reviewed in 10). In contrast to actin microfilaments, microtubules are usually excluded from growth cone filopodia but occasionally extend into select filopodia, possibly stabilizing them as the future track for axonal growth (20). The control of the direction or rate of growth cone motility has direct bearing on axon growth and guidance. Two families of G proteins have been implicated in these events. Manipulation of heterotrimeric G proteins has been shown to affect growth cone morphology and axon outgrowth. The family of ras-related small GTP-binding proteins has been shown to regulate the actin-based cytoskeleton in fibroblasts and may have similar roles in neuronal growth cones.

HETEROTRIMERIC G PROTEINS IN GROWTH CONE MOTILITY AND NEURITE OUTGROWTH Heterotrimeric G proteins are extensively expressed in neurons and have roles in many signal transduction processes. These include the regulation of certain ion channels (reviewed in 21; Dunlap and Ikeda and Haydon and Trudeau, this issue), photoreception in the vertebrate visual system (22) (see Weng, et al., this issue), the b-adrenergic cascade, and many other cellular processes. Establishing these functional roles for the heterotrimeric G proteins has been greatly aided by the application of specific inhibitors and activators of Ga proteins. The inhibitor pertussis toxin selectively inactivates Gai family members by ADPribosylation. ADP-ribosylation blocks the interaction of Ga subunits with their receptors, thereby preventing downstream transmission of the signal by the modified G proteins (23). Conversely, the wasp venom peptide mastoparan activates Gai family members via a receptor-like mechanism (24).

Pertussis toxin and mastoparan have been used in several studies addressing the role of Ga proteins involved in axon outgrowth, sometimes with contradictory results. These may reflect cell type-specific differences in G protein interactions. PC12 cells, a popular model system for studies of neurite outgrowth, are derived from rat pheochromocytomas and, upon exposure to nerve growth factor, extend differentiated processes akin to neurites produced by sympathetic neurons in primary culture (25). PC12 cell neurite outgrowth is enhanced by the neuronal CAMS (cell adhesion molecules) L1, N-CAM, and N-cadherin (26). Treatment of untransformed PC12 cells with ‘‘activating’’ antibodies that bind to these homophilic CAMs changes a variety of second messenger systems (27). Both of these effects are blocked by pertussis toxin (26, 27). Similarly, pertussis toxin inhibits L1-dependent neurite outgrowth from cerebellar neurons cultured on 3T3 cells expressing L1 (28). Conversely, mastoparan increases neurite outgrowth on nontransfected fibroblasts by a pertussis toxin-sensitive mechanism (29). These studies suggest that a Gai family member(s) is involved as a positive regulator of neurite outgrowth, acting downstream of receptors in these cells. In contrast, exposure of embryonic chick dorsal root ganglia (DRG) to mastoparan leads to growth cone collapse that can be blocked by pertussis toxin (30). This suggests that for DRG neurons, a Gai family member may be a negative regulator of growth cone motility. Recent work by Kindt and Lander (31) further complicates this issue. They showed that pertussis toxin inhibits laminin-mediated, chick DRG growth cone guidance as well as growth cone collapse induced by addition of embryonic brain membranes. However, both of these effects are independent of direct G protein involvement. These findings raise the possibility that the interpretation of any experiment using pertussis toxin may be complicated by additional, non-G protein-related effects. Additional data in yet another cell type suggest that at least one Ga protein (though not necessarily a Gai family member) is a negative regulator of axon growth. Introduction of the Ga inactivator GDP b S into chick sympathetic neurons increases neurite extension, whereas introduction of the Ga activator GTPgS decreases it (32). The drug studies described above suggest possible roles for Ga proteins in the regulation of axon outgrowth. They cannot distinguish the individual Ga family members involved. As known targets of pertussis toxin and mastoparan, the Gai family members have been considered the prime suspects. There is

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circumstantial evidence that two Gai family members, Gao and Gai1, may regulate events in early neuronal differentiation, such as axon outgrowth and/or cell body migration. They are abundant in neurites of PC12 cells (33) and in fetal and neonatal rat growth cone membranes (33, 34) as shown by immunocytochemistry, immunoblotting, and cell fractionation studies. Gao expression in the developing nervous system is specifically associated with differentiating, postmitotic neurons, and not with proliferating neuroblasts (35). Evidence that suggests a functional role for Gao in axon growth comes from Strittmatter et al. (36). Transfection of an activated form of Gao into differentiated PC12 and neuroblastoma-derived N1E-115 cells increases total neurite outgrowth, predominantly by increasing the number of neurites per cell. The average length of neurites was unchanged. A subsequent study showed that the Gao-induced increase in neurite number could be blocked either by the protein kinase C (PKC) activator TPA or by thapsigargin, an inhibitor of intracellular calcium release (37). Conversely, PKC inhibitors, such as staurosporine, increased neurite outgrowth with no further increase upon transfection of activated Gao. These results suggest that the neuritestimulatory effects of activated Gao in PC12 cells may involve the inhibition of PKC activity and/or the modulation of intracellular calcium release. This potential Gao regulation of intracellular calcium release is interesting in light of previous evidence that intraneuronal calcium levels are important for axon growth (reviewed in 38). Calcium fluctuations outside of an ‘‘outgrowth permissive range’’ causes a cessation of motility. Gao’s inhibition of plasma membrane, voltagegated calcium channels represents an additional mechanism whereby Gao could regulate intraneuronal calcium levels (39–41) (see Dunlap and Ikeda, this issue). The pharmacological data above tentatively place PKC activity and intracellular calcium levels downstream of Gao. There is in vitro biochemical evidence to suggest that the neuronal growth associated protein GAP-43 may act upstream. In vitro, GAP-43 activates Gao and Gai by stimulating the exchange of GDP for GTP (42). Like Gao, GAP-43 is highly expressed in neuronal growth cones during periods of axon outgrowth (43–46). To assess the functional relationship between GAP-43 and Gao in growth cone motility, we targeted these molecules individually using microscale chromophoreassisted laser inactivation (micro-CALI). CALI (47, 48) and micro-CALI (49) cause acute inactivation of targeted proteins in a spatially restricted manner. It has

been used to demonstrate intracellular roles for a variety of growth cone proteins (50–52). Micro-CALI of GAP-43 in cultured, embryonic chick DRGs causes lamellipodial retraction of the irradiated areas of the growth cones (53). This result is consistent with antisense oligonucleotide studies demonstrating that decreased expression of GAP-43 in cultured chick DRGs leads to unstable lamellipodia (54, 55). Additionally, retinal neurons of transgenic mice deleted for GAP-43 exhibit abnormal pathfinding in the optic chiasm (56). Collectively, these results suggest that GAP-43 regulates the actin-based motility of growth cones, with possible consequences for axon outgrowth and guidance. The hypothesis that Gao is downstream of GAP-43 signaling predicts that their inactivation should cause similar effects in growth cone lamellipodia. As a first step in testing this, CALI of Gao in vitro was shown to cause a 95% inactivation of Gao’s GTPase activity (K. Vancura and D. Jay, unpublished results). Preliminary results from micro-CALI of Gao in embryonic chick DRG growth cones suggest that the functional loss of Gao does not cause lamellipodial retraction in the irradiated region of the growth cones to the extent caused by micro-CALI of GAP-43. Additionally, microCALI of Gao does not significantly affect filopodial motility compared to controls (K. Vancura and D. Jay, unpublished results). Interestingly, transgenic mice with deletions for both isoforms of Gao appear grossly neuroanatomically normal, although they do exhibit tremors and die young (57). Embryonic DRG cultures from these mice appear normal with respect to growth cone collapse in response to brain membrane extract (57). These results do not support a functional role for Gao. Despite its abundance in the growth cone (33, 34) and its ability to stimulate neurite outgrowth in neuronal cell lines (36), direct evidence that Gao functions in growth cone motility or in axon outgrowth in vivo has remained elusive.

SMALL GTP-BINDING PROTEINS AND REGULATION OF THE ACTIN CYTOSKELETON Small GTP-binding proteins have been implicated in cell motility of lamellipodial and filopodial movement in cultured fibroblasts (reviewed in 58). Three members of the rho subfamily (rho, rac, and cdc42) of small GTP-binding proteins, as well as activated ras, have been shown to regulate the formation of filopodia, lamellipodia, actin stress fibers, and focal adhesions in

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cultured fibroblasts (59; reviewed in 60). These findings, together with genetic analysis in Drosophila (61), suggest that small GTP-binding proteins may also act in the actin-based motility of neuronal growth cones and axon growth. The functions of the rho family members have been addressed by microinjection of recombinant forms of these proteins into Swiss 3T3 fibroblasts followed by morphometry and F-actin immunocytochemistry. When activated forms of rho, rac, and cdc42 are injected individually, each results in a distinct reorganization of the actin cytoskeleton. Microinjection of rho leads to the formation of actin stress fibers and focal adhesions (62). Focal adhesions are multimolecular complexes associated with the actin cytoskeleton that contain a number of structural and signal transducing proteins such as src, p125FAK, and b-1 integrins (see 63–66 for reviews). These structures are likely to be important but their precise role in F-actin-based motility remains unclear. When rac is injected, actin-rich lamellipodial ruffles form, followed by the appearance of actin stress fibers (59). Microinjection of activated cdc42 leads to the formation of filopodia, the spreading of lamellipodia, and ultimately, to the formation of actin stress fibers (67, 68). Coinjections of combinations of these same proteins with inhibitors of rac and rho activity have established the functional relationship between the members of the rho subfamily. For example, when a dominant negative form of rac is coinjected with activated cdc42, filopodia still form, but the formation of lamellipodial ruffles and stress fibers is suppressed (67). Coinjection of rac and a rho inhibitor (C3 transferase from Clostridium botulinum) results in lamellipodial ruffling, but stress fibers are not formed (59). Similarly, the ability of activated ras to interact with members of the rho subfamily was demonstrated by injections of activated ras alone or accompanied by rac and rho inactivators into serum-starved Swiss 3T3 cells. The injection of recombinant, activated ras resulted in the formation of lamellipodial ruffles, followed by the appearance of actin stress fibers similar to that induced by injection of rac. These effects were blocked by coinjection of dominant inhibitory rac. Injection of C3 transferase alone blocked stress fiber formation without inhibiting lamellipodial ruffling (59). These experiments reveal that activated ras can act upstream of rac and rho in the formation of lamellipodial ruffles and stress fibers in fibroblasts. However, inhibitors of endogenous ras had no effect on the rac- and rho-mediated cytoskeletal changes of these cells in response to the growth factors PDGF and EGF. Thus, a role for endogenous ras as an

upstream regulator of rac and rho has not been established. The functions of the rho family proteins have continued to be elegantly defined in fibroblasts. Evidence that they also are important regulators of axon outgrowth has begun to accumulate. In a series of experiments using N1E-115 neuroblastoma cells, Moolenaar and colleagues showed that pretreatment with C3 transferase prevents the characteristic neurite retraction induced by exposure of the differentiated cells to serum or thrombin (69, 70). These studies implicate rho as a negative regulator of neurite outgrowth in these cells, but do not identify the site(s) of rho activity within the cell. The preliminary experiments of Kuhn and Bamburg (71) suggest that rho can act in the growth cone to regulate actin polymerization. Injection of either constitutively active or dominant negative forms of rho into primary chick motoneurons greatly reduced neurite elongation without affecting neurite initiation or neuronal adhesion. Immunocytochemistry revealed that in both cases, the F-actin distribution in the growth cone was perturbed. Neurons loaded with constitutively active rho accumulated excessive levels of F-actin, whereas those loaded with dominant negative rho were depleted of it. The data discussed thus far addresses the function of rho and ras family proteins in cultured cells. Evidence that members of the rho subfamily function in axon outgrowth in vivo comes from the work of Luo et al. (61). They examined the expression and function of the Drosophila homologues of rac and cdc42 in the embryonic nervous system. The cDNAs encoding DracI and Dcdc42, the Drosophila homologues of rac and cdc42, were cloned and found to be .90% identical to their human counterparts at the amino acid level. In situ hybridization revealed that DracI transcripts are expressed in the embryonic nervous system at stage 13, the period of axonogenesis, and persist at least through stage 16. A similar pattern of expression was observed for Dcdc42. Luo et al. (61) addressed in vivo neuronal function by expressing constitutively active and dominant negative forms of these G proteins using a series of nervous system-specific promoters in Drosophila embryos. The expressed proteins resulted in varying degrees of embryonic lethality, with defects in axon outgrowth and myoblast fusion. The effect on axon outgrowth was examined for the peripheral nervous system neurons of the dorsal and lateral clusters. Neurons in the dorsal clusters of each segment normally project to

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neurons in the lateral clusters. Early onset expression (stage 10/11) of either constitutively active or dominant negative DracI interfered with these projections in 70% of the segments examined and caused axon loss in the most severe case. With a later onset of expression (stage 12), axons were often found ‘‘stalled’’ between the dorsal and lateral clusters, suggesting that DracI is required for axon elongation as well as initiation. Interestingly, the dendrites of dorsal cluster neurons were unaffected in these experiments. In contrast, when the dominant negative form of Dcdc42 was expressed in parallel experiments, both axonal and dendritic projections were perturbed, and the position of the neurons within the clusters was also altered. This suggests that individual rho family members may regulate distinct aspects of neuronal morphogenesis. The apparent paradox that both constitutively active and dominant negative forms of DracI resulted in similar phenotypes was partially resolved by the examination of the patterns of immunofluorescent localization of F-actin in the mutant embryos. F-actin accumulates abnormally in dorsal neuron clusters of embryos expressing constitutively active DracI, but not in those expressing the dominant negative form. This finding suggests that constitutively active and dominant inhibitory forms of DracI may produce their similar morphological defects by different mechanisms. While the localization of F-actin in individual cells was not determined in this study, it appears likely, in light of the strong evidence discussed above for fibroblasts, that the actin cytoskeleton is a key target for regulation by rho family members during axonal outgrowth. Work in both fibroblast and neuronal cell types has led to the ordering of the following pathway: activation of cdc42 leads to the formation of filopodia and to the stimulation of rac. Ras can also activate rac, which in turn gives rise to the formation of lamellipodia and the activation of rho. Active rho then causes the formation of actin-dominated stress fibers and focal adhesions in fibroblasts and neurite retraction in neuronal cells. Figure 1 summarizes these results by speculating that a similar pathway may regulate growth cone motility and axon outgrowth.

AXON GROWTH AND VESICULAR TRANSPORT Axon growth requires an enormous investment on the part of the cell, as it must construct and extend a neurite process many times longer than the cell body.

Of particular importance is the requirement of vesicular transport during axon extension. Vesicular transport is essential for axon growth. It has been estimated that a single neurite elongating 1 mm per day in culture requires the addition of ,1 µm2 of new membrane per minute (72). Continued axon elongation depends on the anterograde transport of membrane proteins synthesized in the soma (73, 74). Furthermore, the need for integrating axon growth within the context of the extracellular environment makes it a highly regulated process. Recent studies have implicated small GTP-binding proteins as important components of the regulatory mechanisms that govern axon outgrowth by controlling vesicular transport.

SMALL GTP-BINDING PROTEINS AND VESICULAR TRANSPORT In the neuron, as in every cell type examined so far, vesicular traffic is highly regulated, and members of the rab subfamily of ras-related small GTP-binding proteins are key players. The rab subfamily of proteins contains over 30 members. They are ubiquitously expressed; many of the members have been found in every cell type examined so far (75). The first indication that rab family members control vesicular transport came from studies of the related yeast protein Sec4p. Loss of function mutations in Sec4p result in an accumulation of vesicular traffic in the Golgi apparatus (76). The exact roles of the rab family members remain unclear. Cellular transport of membrane vesicles can be divided into four steps: vesicle budding, targeting, motor dependent movement, and fusion. In principal, any one or all of these steps could be regulated by the rab family. A current model for rab protein function is as follows: the rab protein is recruited to nascent vesicles in the GDP-bound form, and the exchange of GDP for GTP accompanies vesicle budding. The GTPbound form is associated with the vesicle as it translocates, and GTP hydrolysis may be a switch that triggers vesicle fusion and the recycling of the rab protein (4, 75). Perhaps the most striking feature of the rab family of molecules is the remarkable specificity of their localization and function (4, 75). Individual rab proteins are localized to and regulate distinct vesicular traffic routes within the cell. Rab2 localizes to and controls traffic between the endoplasmic reticulum (ER) and the Golgi, rab5a regulates traffic from the plasma membrane to early endosomes, and rab8 controls transport

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from the trans Golgi to the cell surface. These three proteins, rab2, 5a, and 8, together with rab3a, which is specific for synaptic vesicles, are the family members for which the most is known with regards to their function in neurons. They will be considered in more detail below. Rab8 is perhaps the best characterized rab protein with respect to neuronal expression and function. In hippocampal neurons cultured in vitro, rab8 is found initially in every neurite (77). As hippocampal neurons mature in vitro, one neurite eventually differentiates into an axon and the remainder into dendrites; rab8 expression becomes restricted to the soma and the dendrites at this time (78). Injection of antisense oligonucleotides against rab8 decreases the transport of a marker protein from the Golgi to the dendritic plasma membrane of mature hippocampal neurons (78). In immature hippocampal neurons, these oligonucleotides block morphological maturation and process outgrowth (77). Videoenhanced differential interference contrast microscopy revealed that anterograde transport of vesicles in the neurites of treated cells appears to be inhibited. This result was confirmed with the use of the fluorescent marker bodipy-ceramide. Because the emission wavelength of bodipy-ceramide varies with the molar density of membrane lipid, it can be used as a marker for newly formed exocytic vesicles (79). Since different organelles have characteristic lipid compositions, it is possible to identify the point of blockage from bodipyceramide’s emission wavelength. In untreated neurons, bodipy-ceramide labels the neurites and the Golgi, while in neurons treated with the rab8 antisense oligos, only the Golgi is labeled (77). These results identify rab8 as a key regulator of anterograde vesicle traffic in differentiating neurons and further suggest that rab8 functions at an early step (such as vesicle formation, budding, or binding to microtubules). The possibility that rab8 also functions later, e.g., in vesicle fusion, cannot be ruled out, however. During neuronal differentiation, competition for target-derived trophic factors is thought to be a key regulatory mechanism for process outgrowth and synapse formation. Trophic factors must be efficiently internalized and transported from the growth cone to the soma to exert their effects. The experiments of de Hoop et al. (80) have identified rab5a as a likely regulator of this important process. The rab5a protein has been previously localized to the plasma membrane, clathrin-coated vesicles, and early endosomes in nonneuronal cells (81). It is found on both the dendrites and the axon of cultured hippocampal neu-

rons. The amount of rab5a, as determined by immunoblotting analysis, increases as these processes elongate and mature. Overexpression of either a wild-type or a GTPase-deficient form of rab5a, that may be constitutively active, results in enlarged endosomes. This was observed in both the axonal and the somatodendritic regions. These experiments implicate rab5a as an important regulator of endocytosis in neurons. Interestingly, the rab5a was also found on the synaptic vesicles of hippocampal neurons suggesting that synaptic vesicle recycling may be accomplished via endosomes in these neurons. Studies of the rab3a protein have added to our understanding of synaptic vesicle formation and function. Rab3a is an unusual member of the rab subfamily in that its expression is tissue specific. Rab3a is restricted to the nervous system, where it is found on synaptic vesicles (82). The fusion of synaptic vesicles with the cell membrane is accompanied by release of rab3a from the vesicles (82, 83). These findings suggest that rab3a may regulate this process. This is supported by studies using mast cells that express closely related members of the rab3 class. Injection of a peptide corresponding to the rab3 effector domain into mast cells results in the rapid fusion of the entire cellular complement of exocytic granules (84). Conversely, antisense oligonucleotides to rab3b were found to inhibit regulated secretion in pituitary cells, without perturbing endocytosis (85). Together, these experiments suggest that rab3a is likely to function in the pathway that triggers synaptic vesicle fusion. Whether rab3a has a functional role in neurons prior to synapse formation is less clear. Huber et al. (77) dramatically reduced rab3a expression in immature hippocampal neurons using antisense oligonucleotides, and this did not have any obvious consequence for neurite outgrowth. The subcellular localization of individual rab proteins has provided reliable clues as to their functions in synapse formation. However, it should be noted that these proteins may also function globally and affect axon growth nonspecifically. For example, injection of activated rab2 regulates traffic from the ER to the Golgi (81). In dissociated, embryonic rat brain neurons, this results in increased spreading, adhesion, and neurite outgrowth (86). The mechanism by which this occurs remains unclear. Perhaps any enhancement of vesicular flow toward the plasma membrane would have a similar effect. This result raises the possibility that neurite extension could be enhanced by increasing membrane flow toward the cell surface. As the rab family is important for the regulation of vesicular

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traffic, it seems reasonable to speculate that rab proteins themselves are subject to regulation that ensures the integration of intracellular membrane traffic into the overall scheme of cellular function and differentiation (e.g., 4).

DISCUSSION AND FUTURE DIRECTIONS A great deal of progress has been attained recently in our understanding of how G proteins act in axon growth. This has benefited from work in nonneuronal systems and has been aided by the application of a number of different and complementary approaches including pharmacological perturbation, expression of recombinant molecules, and knockout techniques. Major challenges remain for this field. It is necessary to identify the components of G protein-mediated pathways. To understand how G proteins control axon growth, we need to identify upstream components that couple to membrane receptors and downstream components that interact with the cytoskeleton. We must begin to confront the complexity of the multiple pathways that must integrate to control axon growth. Addressing these challenges will help us to better understand the cellular basis of neural development and regeneration. Use of pertussis toxin and mastoparan has greatly advanced our general understanding of heterotrimeric G protein function, but their inclusion in studies of neurite outgrowth has yielded conflicting results. Despite these contradictory results, it is clear that Ga proteins regulate axon outgrowth, although a role in growth cone motility per se has not been clearly demonstrated. This could be due, in part, to cell type-specific differences in G protein function. It may also be a consequence of the fact that each drug affects multiple proteins, a fact that has also limited our ability to ascribe specific functions to individual G proteins. The uncoupling of the multiple pathways of G protein-mediated signal transduction remains elusive. The use of specific perturbation techniques, such as genetic knockout, CALI, and antisense oligonucleotide injection, may provide powerful, complementary strategies to identify the roles of these proteins in axon outgrowth. In contrast to the heterotrimeric G proteins whose functional roles remain largely undefined, addressing the roles of the small GTP-binding proteins of the rho and rab subfamilies has been more fruitful. The application of recombinant, constitutively active, and dominant-inhibitory forms of these proteins has rapidly

advanced our understanding of their functions and their functional relationships to one another. Future work will identify components of these pathways that link signal transduction to cytoskeletal dynamics. One of these studies has eliminated a possible effector of the G proteins to help narrow down the possibilities (87)—p65PAK is not downstream of rac and cdc42 in the induction of lamellipodia and filopodia in fibroblasts. This field will continue to benefit from work in nonneuronal systems and molecular approaches. The conservation of individual G proteins throughout evolution suggests that use of the two hybrid screens will be useful. Degenerate PCR approaches may identify other G protein families that function in axon growth. For members of the rab subfamily, the availability of an in vitro model system in which neuronal polarity is reliably recapitulated (i.e., cultured hippocampal neurons) has facilitated rapid progress in elucidating the functions of rab family proteins in neurons. This field is currently active in addressing functional interactions and finding other components of rab-mediated pathways. One such protein is Rab3A GDI, which can regulate GDP-bound rab3a’s dissociation from membranes (88, 89). Further experiments capitalizing on data obtained in nonneuronal systems should also identify other targets involved in axon growth. For example, in transfected mammalian 293T cells, GTPasedeficient rab8 interacts with the serine/threonine kinase rab8ip (90). For the rho subfamily, most of the experiments to date have been done in fibroblasts, and the functions of the family members in neurons are just beginning to be elucidated. The preliminary experiments of Kuhn and Bamburg (71), in which rho was found to regulate the accumulation of actin in the growth cone, suggest that at least some of the findings from fibroblasts will also be applicable to neuronal growth cones. The work of Luo et al. (61), in which mutant forms of the Drosophila homologues DracI and Dcdc42 were expressed in vivo, shows the power of genetic approaches to identify upstream and downstream effectors in the rho family signaling pathways. This study also serves as a cautionary example of how simple functional relationships observed in cultured cells can be obscured by the more complex in vivo environment. Dominant inhibitory and constitutively active forms of DracI expressed in the embryo superficially caused similar defects in the developing nervous system. Similar results were obtained for rho in cultured neurons (71). Together these findings suggest that a balance of G protein signaling is required for normal axon growth. This is not surprising given

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the diversity of processes that must be regulated during axon growth. This is analogous to the ‘‘set point’’ hypothesis for calcium regulation of axon growth (38). This balance may be a general requirement for the dynamic equilibria necessary for the maintenance and integration of cellular processes. A deeper analysis of these mechanisms will serve us well in understanding G protein function in the vertebrate nervous system in vivo.

ACKNOWLEDGMENTS The authors thank Eva Neer for providing a preprint of her work and Christopher Stipp and Paul Diamond for critical reading. KLV was supported in part by a training grant from the NIH. DGJ was a John L. Loeb Associate Professor and is supported by NIH and the Klingenstein Foundation.

REFERENCES 1. Neer, E. J. (1995) Heterotrimeric G proteins: Organizers of transmembrane signals. Cell 80, 249–257. 2. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh, T. (1991) Structure and function of signal-transducing GTPbinding proteins. Annu. Rev. Biochem. 60, 349–400. 3. Hall, A. (1994) Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell. Biol. 10, 31–54. 4. Nuoffer, C., and Balch, W. E. (1994) GTPases: Multifunctional molecular switches regulating vesicular traffic. Annu. Rev. Biochem. 63, 949–990. 5. Jessell, T. M. (1991) Cell migration and axon guidance. In Principles of Neural Science (E. R. Kandel, J. H. Schwartz, and T. M. Jessell, Eds.), pp. 908–928. Appleton & Lange, Norwalk, CT. 6. Davenport, R. W., Dou, P., Rehder, V., and Kater, S. B. (1993) A sensory role for neuronal growth cone filopodia. Nature 361, 721–724. 7. Tanaka, E., and Sabry, J. (1995) Making the connection: Cytoskeletal rearrangements during growth cone guidance. Cell 83, 171–176. 8. Bray, D., and Chapman, K. (1985) Analysis of microspike movements on the neuronal growth cone. J. Neurosci. 5, 3204– 3213. 9. Goldberg, D. J., and Burmeister, D. W. (1986) Stages in axon formation: Observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy. J. Cell Biol. 103, 1921–1931. 10. Gordon-Weeks, P. R. (1993) Organization of microtubules in axonal growth cones: a role for microtubule-associated protein MAP1B. J. Neurocytol. 22, 717–725. 11. Letourneau, P. C., Kater, S. B., and Macagno, E. R. (1992) The Nerve Growth Cone. Raven Press, New York. 12. Bandtlow, C., Zachleder, T., and Schwab, M. E. (1990) Oligodendrocytes arrest neurite growth by contact inhibition. J. Neurosci. 10, 3837–3848. 13. O’Connor, T. P., Duerr, J. S., and Bentley, D. (1990) Pioneer growth cone steering decisions mediated by single filopodial contacts in situ. J. Neurosci. 10, 3935–3946.

14. Bovolenta, P., and Mason, C. (1987) Growth cone morphology varies with position in the developing mouse visual pathway from retina to first targets. J. Neurosci. 7, 1447–1460. 15. Taghert, P. H., Bastiani, M. J., Ho, R. K., and Goodman, C. S. (1982) Guidance of pioneer growth cones: Filopodial contacts and coupling revealed with an antibody to Lucifer Yellow. Dev. Biol. 94, 391–399. 16. Tosney, K. W., and Landmesser, L. T. (1985) Growth cone morphology and trajectory in the lumbosacral region of the chick embryo. J. Neurosci. 2345–2348. 17. Eisen, J. S., Myers, P. Z., and Westerfield, M. (1986) Pathway selection by growth cones of identified motoneurons in live zebra fish embryos. Nature 320, 269–271. 18. Holt, C. E. (1989) A single-cell analysis of early retinal ganglion cell differentiation in Xenopus: From soma to axon tip. J. Neurosci. 9, 3123–3145. 19. Letourneau, P. C. (1983) Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J. Cell. Biol. 97, 963–973. 20. Sabry, J. H., O’Connor, T. P., Evans, L., Toroian, R. A., Kirschner, M., and Bentley, D. (1991) Microtubule behavior during guidance of pioneer neuron growth cones in situ. J. Cell Biol. 115, 381–395. 21. Hille, B. (1992) G protein-coupled mechanisms and nervous signaling. Neuron 9, 187–195. 22. Stryer, L. (1991) Visual excitation and recovery. J. Biol. Chem. 266, 10711–10714. 23. Neer, E. J. (1994) G proteins: Critical control points for transmembrane signals. Protein Sci. 3, 3–14. 24. Higashijima, T., Burnier, J., and Ross, E. M. (1990) Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. J. Biol. Chem. 265, 14176–14186. 25. Greene, L. A., and Tischler, A. S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424–2428. 26. Doherty, P., Ashton, S. V., Moore, S. E., and Walsh, F. S. (1991) Morphoregulatory activities of NCAM and N-Cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca21 channels. Cell 67, 21–33. 27. Schuch, U., Lohse, M. J., and Schachner, M. (1989) Neural cell adhesion molecules influence second messenger systems. Neuron 3, 13–20. 28. Williams, E. J., Doherty, P., Turner, G., Reid, R. A., Hemperly, J. J., and Walsh, F. S. (1992) Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1. J. Cell Biol. 119, 883–892. 29. Williams, E. J., Walsh, F. S., and Doherty, P. (1994) Tyrosine kinase inhibitors can differentially inhibit integrin-dependent and CAMstimulated neurite outgrowth. J. Cell Biol. 124, 1029–1037. 30. Igarashi, M., Strittmatter, S. M., Vartanian, T., and Fishman, M. C. (1993) Mediation by G proteins of signals that cause collapse of growth cones. Science 259, 77–79. 31. Kindt, R. M., and Lander, A. D. (1995) Pertussis toxin specifically inhibits growth cone guidance by a mechanism independent of direct G protein inactivation. Neuron 15, 79–88. 32. Strittmatter, S. M., Vartanian, T., and Fishman, M. C. (1992) GAP-43 as a plasticity protein in neuronal form and repair. J. Neurobiol. 23, 507–520. 33. Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Go is a major growth cone protein subject to regulation by GAP-43. Nature 344, 836–841.

Copyright r 1998 by Academic Press

218

Vancura and Jay

34. Edmonds, B. T., Moomaw, C. R., Hsu, J. T., Slaughter, C., and Ellis, L. (1990) The p38 and p34 polypeptides of growth cone particle membranes are the a- and b-subunits of G proteins. Dev. Brain Res. 56, 131–136. 35. Schmidt, C. J., Zubiaur, M., Valenzuela, D., Neer, E. J., and Drager, U. C. (1994) Go, a guanine nucleotide binding protein, is expressed during neurite extension in the embryonic mouse. J. Neurosci. Res. 38, 182–187. 36. Strittmatter, S. M., Fishman, M. C., and Zhu, X.-P. (1994) Activated mutants of the a subunit of Go promote an increased number of neurites per cell. J. Neurosci. 14, 2327–2338. 37. Xie, R., Li, L., Goshima, Y., and Strittmatter, S. M. (1995) An activated mutant of the a subunit of Go increases neurite outgrowth via protein kinase C. Dev. Brain Res. 87, 77–86. 38. Kater, S. B., and Mills, L. R. (1991) Regulation of growth cone behavior by calcium. J. Neurosci. 11, 891–899. 39. Ewald, D. A., Sternweiss, P. C., and Miller, R. J. (1988) Guanine nucleotide binding protein Go-induced coupling of neuropeptide Y receptors to Ca21 channels in sensory neurons. Proc. Natl. Acad. Sci. USA 85, 3633–3637. 40. Hescheler, J., Rosenthal, W., Trautwein, W., and Schultz, G. (1987) The GTP-binding protein, Go, regulates neuronal calcium channels. Nature 325, 445–447. 41. Kleuss, C., Heschler, J., Ewel, C., Rosenthal, W., Schultz, G., and Wittig, B. (1991) Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium channels. Nature 353, 43–48. 42. Strittmatter, S. M., Valenzuela, D., Sudo, Y., Linder, M. E., and Fishman, M. C. (1991) An intracellular guanine nucleotide release protein for Go: GAP-43 stimulates isolated a subunits by a novel mechanism. J. Biol. Chem. 266, 22465–22471. 43. Meiri, K. F., Pfenninger, K. H., and Willard, M. (1986) Growth associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46 a major polypeptide of a subcellular fraction that is enriched in growth cones. Proc. Natl. Acad. Sci. USA 83, 3537–3541. 44. Meiri, K. F., Willard, M., and Johnson, M. I. (1988) Distribution and phosphorylation of the growth associated protein GAP-43 in regenerating sympathetic neurons in culture. J. Neurosci. 8, 2571–2581. 45. Skene, J. H. P., Jacobson, R. D., Snipes, G. J., MacGuire, C. B., Norden, J., and Freeman, J. A. (1986) A protein induced during nerve growth, GAP-43 is a major component of growth cone membranes. Science 233, 783–785. 46. Goslin, K., Schreyer, D. J., Skene, J. H., and Banker, G. A. (1988) Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature 336, 672–674. 47. Jay, D. G. (1988) Selective destruction of protein function by chromophore-assisted laser inactivation. Proc. Natl. Acad. Sci. USA 85, 5454–5458. 48. Jay, D. G., and Keshishian, H. (1990) Laser inactivation of fasciclin I disrupts axon adhesion of grasshopper pioneer neurons. Nature 348, 548–550. 49. Diamond, P., Mallavarapu, A., Schnipper, J., Booth, J., Park, L., O’Connor, T. P., and Jay, D. G. (1993) Fasciclin I and II have distinct roles in the development of grasshopper pioneer neurons. Neuron 11, 1–20. 50. Chang, H. Y., Takei, K., Sydor, A. M., Born, T., Rusnak, F., and Jay, D. G. (1995) Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin. Nature 376, 868–690.

51. Wang, F., Wolenski, J. S., Cheney, R. E., Mooseker, M. S., and Jay, D. G. (1996) Function of Myosin-V in filopodial extension of neuronal growth cones. Science 273, 660–663. 52. Sydor, A. M., Su, A. L., Wang, F.-S., Xu, A., and Jay, D. G. (1996) Talin and vinculin functions have distinct roles in filopodial motility in the neuronal growth cone. J. Cell Biol. 134, 1197–1207. 53. Vancura, K., Wang, P., Lee, G., and Jay, D. G. (1995) Tau and GAP-43 are potential downstream effectors of calcineurin during growth cone motility. Am. Soc. Cell. Biol. Annu. Meeting Abstr. 54. Aigner, L., and Caroni, P. (1993) Deletion of 43-kD growthassociated protein in primary sensory neurons leads to diminished formation and spreading of growth cones. J. Cell Biol. 123, 417–429. 55. Aigner, L., and Caroni, P. (1995) Absence of persistent spreading, branching, and adhesion in GAP-43 depleted growth cones. J. Cell Biol. 128, 647–660. 56. Strittmatter, S. M., Fankhauser, C., Huang, P. L., Mashimo, H., and Fishman, M. C. (1995) Neuronal pathfinding is abnormal in mice lacking the neuronal growth cone protein GAP-43. Cell 80, 445–452. 57. Valenzuela, D., Han, X., Mende, U., Fankhouser, C., Mashimo, H., Huang, P., Pfeffer, J., Neer, E. J., and Fishman, M. C. (1997) Gao is necessary for muscarinic regulation of Ca11 channels in mouse heart. Proc. Natl. Acad. Sci. USA 94, 1727–1732. 58. Nobes, C. D., and Hall, A. (1995b) Rho, rac, & cdc42 GTPases: Regulators of actin structures, cell adhesion and motility. Biochem. Soc. Transact. 23, 456–459. 59. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410. 60. Chant, J., and Stowers, L. (1995) GTPase cascades choreographing cellular behavior: Movement, morphogenesis, and more. Cell 81, 1–4. 61. Luo, L., Liao, Y. J., Jan, L. Y., and Jan, Y. N. (1994) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8, 1787–1802. 62. Ridley, A. J., and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399. 63. Burridge, K., Fath, K., Kelly, T., Nuckolis, G., and Turner, C. (1988) Focal adhesions: Transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 4, 487–525. 64. Gumbiner, B. M. (1993) Proteins associated with the cytoplasmic surface of adhesion molecules. Neuron 11, 551–564. 65. Turner, C. E., and Burridge, K. (1991) Transmembrane molecular assemblies in cell–extracellular matrix interactions. Curr. Opin. Cell. Biol. 3, 849–853. 66. Sastry, S. K., and Horwitz, A. F. (1993) Integrin cytoplasmic domains: Mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr. Opin. Cell. Biol. 5, 819–831. 67. Nobes, C. D., and Hall, A. (1995a) Rho, rac, & cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62. 68. Kozma, R., Ahmed, S., Best, A., and Lim, L. (1995) The Rasrelated protein cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15, 1942–1952.

Copyright r 1998 by Academic Press

219

Axon Growth 69. Jalink, K., and Moolenaar, W. H. (1992) Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers. J. Cell Biol. 118, 411–419. 70. Jalink, K., van Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H. (1994) Inhibition of lysophosphatidateand thrombin-induced neurite retraction and neuronal cell rounding by ADP Ribosylation of the small GTP-binding protein rho. J. Cell Biol. 126, 801–810. 71. Kuhn, T. B., and Bamburg, J. R. (1996) The small GTPase rac is essential for the formation and differentiation of neurites from motorneurons mediated by extracellular matrix molecules. Am. Soc. Cell. Biol. Annu. Meeting Abstr. 72. Pfenninger, K. H., and Maylie-Pfenninger, M. F. (1981) Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J. Cell Biol. 89, 547–559. 73. Craig, A. M., Wyborski, R. J., and Banker, G. (1995) Preferential addition of newly synthesized membrane protein at axonal growth cones. Nature 375, 592–594. 74. Dai, J., and Sheetz, M. P. (1995) Axon membrane flows from the growth cone to the cell body. Cell 83, 693–701. 75. Simons, K., and Zerial, M. (1993) Rab proteins and the road maps for intracellular transport. Neuron 11, 789–799. 76. Salminen, A., and Novick, P. J. (1987) A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 49, 527–538. 77. Huber, L. A., Dupree, P., and Dotti, C. G. (1995) A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons. Mol. Cell. Biol. 15, 918–924. 78. Huber, L. A., De Hoop, M. J., Dupree, P., Zerial, M., Simons, K., and Dotti, C. (1993) Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p. J. Cell Biol. 123, 47–55. 79. Pagano, R. E., Martin, O. C., Kang, H. C., and Haugland, R. P. (1991) A novel fluorescent ceramide analog for studying membrane traffic in animal cells: Accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J. Cell Biol. 113, 1267–1279. 80. De Hoop, M. J., Huber, L. A., Stenmark, H., Williamson, E., Zerial, M., Parton, R. G., and Dotti, C. G. (1994) The involvement

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

of the small GTP-binding protein rab5a in neuronal endocytosis. Neuron 13, 11–22. Chavrier, P., Parton, R. G., Hauri, H. P., Simons, K., and Zerial, M. (1990) Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 62, 317– 329. Fischer von Mollard, G., Mignery, G. A., Baumert, M., Perin, M. S., Hanson, T. J., Burger, P. M., Jahn, R., and Sudhof, T. C. (1992) Rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc. Natl. Acad. Sci. USA 87, 1988–1992. Matteoli, M., Takei, K., Cameron, R., Hurlbut, P., Johnston, P. A., Sudhof, T. C., Jahn, R., and De Camilli, P. (1991) Association of rab3a with synaptic vesicles at late stages of the secretory pathway. J. Cell Biol. 115, 625–633. Oberhauser, A. F., Monck, J. R., Balch, W. E., and Fernandez, J. M. (1992) Exocytic fusion is activated by rab3a peptides. Nature 360, 270–273. Lledo, P. M., Vernier, P., Vincent, J. D., Mason, W. T., and Zorec, R. (1993) Inhibition of rab3b expression attenuates Ca21-dependent exocytosis in rat anterior pituitary cells. Nature 364, 540–544. Ayala, J., Touchot, N., Zahraoui, A., Tavitian, A., and Prochiantz, A. (1990) The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro. Neuron 4, 797–805. Lamarche, N., Tapon, N., Stowers, L., Burbelo, P. D., Aspenstrom, P., Bridges, T., Chant, J., and Hall, A. (1996) Rac and cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell 87, 519–525. Araki, S., Kikuchi, A., Hata, Y., Isomura, M., and Takai, Y. (1990) Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J. Biol Chem. 265, 13007–13015. Regazzi, R., Kikuchi, A., Takai, Y., and Wollheim, C. B. (1992) The GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J. Biol. Chem. 267, 17512–17519. Ren, M., Zeng, J., De Lemos-Chiarandini, C., Rosenfeld, M., Adesnik, M., and Sabatini, D. D. (1996) In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase. Proc. Natl. Acad. Sci. USA 93, 5151–5155.

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