Axon guidance: the cytoplasmic tail

221

Axon guidance: the cytoplasmic tail Bharatkumar N Patel* and David L Van Vactor† Recent advances in the study of axon guidance have begun to clarify the intricate signalling mechanisms utilised by receptors that mediate path-finding. Many of these axon guidance receptors, including Plexin B, EphA, ephrin B and Robo, regulate the Rho family of GTPases, to effect changes in motility. Recent studies demonstrate a critical role for the cytoplasmic tails of guidance receptors in signalling and also reveal the potential for a great deal of crosstalk between the various receptor-signalling pathways. Addresses Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA *e-mail: [email protected] † e-mail: [email protected] Correspondence to: David L Van Vactor Current Opinion in Cell Biology 2002, 14:221–229 0955-0674/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 January 2002 DOI 10.1016/S0955-0674(02)00308-3 Abbreviations Abi-1 Abl-interacting protein-1 CAP Cbl-associated protein DCC deleted in colorectal cancer GAP GTPase-activating protein GEF guanine nucleotide exchange factor PDZ PSD-95/Dlg/ZO-1 RGS regulator of G-protein signalling Robo roundabout SH2/3 Src homology 2/3 SrGAP Slit–Robo GAP

Introduction Neurons in the developing central nervous system must make contacts with targets that may be thousands of celldiameters away. The axon has to navigate through a terrain consisting of various cell types, neuronal processes and extracellular matrix molecules. Within this complex terrain are guidance cues that direct the growth cone, the motile tip of the axon, to its target. These cues consist of molecules that attract the extending axon to the appropriate target as well as molecules that repel it from inappropriate pathways. A large number of the molecular cues and their receptors that guide axons to their targets have been identified in different systems and in various organisms [1]. Among the most important families of receptors that regulate axon guidance are the receptor tyrosine kinases [2,3], the receptor tyrosine phosphatases [4], the immunoglobulin family of cell adhesion molecules [5], the cadherins [6], the plexins and neuropilins [7,8], the netrin receptors [9] and the roundabout (Robo) family of receptors ([10]; Figure 1). With a large number of the receptors and their ligands identified, the focus has turned to examining the intracellular

signalling mechanisms that transduce the signals at the cell surface into changes in growth cone motility. As the signalling pathways utilised by the individual guidance cues come into focus, the question of how the growth cone integrates the multiplicity of signals to produce the coordinated changes in cytoskeletal dynamics can be addressed. One of the emerging themes from the recent work is the major importance of the cytoplasmic domains of guidance receptors in signalling and the diverse mechanisms utilised by the cytoplasmic domains to regulate axon guidance. Because of space limitations, this review will focus primarily on major developments within the past year for a limited number of receptors whose signalling reveals a common theme of regulating GTPases to effect changes in motility. The reader is referred to the excellent reviews cited for further information on other receptor families.

Receptor regulation of GTPases The importance of the Rho family of GTPases (including Cdc42, Rac and RhoA) in regulating cell motility and axon guidance is well established [11]. Investigations in many different systems indicate that attraction is mediated primarily by the Cdc42 and Rac GTPases, whereas repulsion is mediated by the Rho GTPases ([11]; Figure 2). In neuronal cell lines, activation of RhoA stimulates actinomysin contractility and stress fibre formation and leads to growth cone collapse. In contrast, activation of Cdc42 and Rac results in the extension of filopodia and lamellipodia, respectively [12]. The Rho GTPases are regulated by guanine nucleotide exchange factors (GEFs), which stimulate the GTPases by promoting GDP release, and by GTPaseactivating proteins (GAPs), which inhibit the GTPases by promoting GTP hydrolysis [11]. By modulating the balance of activation of these GTPases, guidance cues can determine whether a growth cone is attracted or repelled [13]. Indeed, recent work demonstrates that a large number of guidance receptors regulate the Rho GTPases to effect changes in motility. Plexins

Among the axon guidance receptors that regulate the Rho GTPases are the plexins, a family of receptors for the semaphorins — a large group of membrane-anchored and secreted axon-guidance molecules [7,8]. The plexins are required to mediate the growth cone collapsing activity of the semaphorins. The highly conserved and unique sex-plexin domain, found in the large cytoplasmic tail of the receptors, is likely to trigger the signalling cascade leading to growth cone collapse [14]. Indeed, the Plexin B cytoplasmic domain has been found previously to bind Rac directly [15,16]. A recent report from Goodman and colleagues [17•] now suggests that Plexin B mediates motor axon guidance by

222

Cell regulation

Figure 1

Example

Selected members Extracellular

Some of the major families of axon guidance molecules as illustrated by representative members.

Intracellular

Robo1,2,3 (Drosophila)

Robo1

DCC

DCC (mammalian); Frazzled (Drosophila); UNC-40 (C.elegans)

Plexin B

Plexins A-D (mammalian); Plexins A,B (Drosophila)

DN-Cadherin

DN-Cadherin, DE-Cadherin (Drosophila); E-,N-,P-,R-,VECadherin (mammalian)

NCAM

NCAM, L1, TAG, VCAM (mammalian)

LAR

LAR, PTPσ, PTPδ (mammalian); DLAR, DPTP69D (Drosophila)

ephrinB

ephrin B1-B3 (mammalian)

ephrinA

ephrin A1-A5 (mammalian)

EphA

EphA1-A8, EphB1-B6 (mammalian)

Ig loop

Cadherin domain

MRS domain

Fibronectin type III domain

β-catenin domain

G-P/IPT motif

Tyrosine kinase domain

Sema domain

P repeat

Phosphatase domain

Sex-plexin domain

Robo CC domain

Globular domain

Cysteine-rich domain Current Opinion in Cell Biology

inhibiting active Rac and enhancing RhoA signalling in Drosophila. The researchers found that Plexin B directly interacts with Rac in vitro and, at high molar excess, inhibits Rac’s ability to associate with and activate its downstream effector, the serine/threonine kinase PAK1, by activating LIM kinase and inhibiting myosin light chain kinase, which limits depolymerisation and the retrograde flow of actin filaments [18,19]). The biochemical experiments also demonstrated that Plexin B binds to and activates RhoA. Although the biochemical interactions demonstrated

in vitro have not been confirmed in vivo, dosage-sensitive genetic interactions between Plexin B and the GTPases suggest that Plexin B mediates repulsion by inactivating Rac and activating RhoA (Figure 3). Because a Plexin B loss-of-function mutant is not yet available, future experiments may address some remaining issues, including clarifying the role of Plexin B in vivo through an analysis of loss-offunction mutants and examining whether Plexin A, which also plays a role in motor axon guidance in Drosophila [7,8,20], signals in a similar manner.

Axon guidance: the cytoplasmic tail Patel and Van Vactor

223

Figure 2 Model illustrating the role of the Rho GTPases in axon guidance. Attractive cues generally activate Rac and Cdc42 while inactivating RhoA. In contrast, repulsive cues generally inhibit Rac and Cdc42 while activating RhoA. Note that individual attractive and repulsive cues may not affect all of the Rho GTPases simultaneously. It is likely that the balance of activation of the Rho GTPases rather than the activation state of any particular member affects whether a growth cone is attracted or repelled to a guidance cue. Evidence for the effector pathways downstream of the Rho GTPases comes primarily from in vitro studies on non-neuronal and neuronal cells. Arp2/3, actin-related protein 2/3; CRMP, collapsin response mediator protein; LIMK, LIM kinase; MLCK, myosin light chain kinase; N-WASP, neuronal Wiskott–Aldrich syndrome protein; ROCK, Rho-associated kinase; RLC, myosin regulatory light chain.

Attractive cues

Repulsive cues

Cdc42-GTP

Rac-GTP

RhoA-GTP

PAK1

MLCK

LIMK

N-WASP

ROCK

RLC

cofilin

Arp2/3

CRMP

Decreased retrograde actin flow

Decreased actin depolymerization

Increased actin nucleation

?

Increased retrograde actin flow

Increased actin depolymerization

Decreased actin nucleation

?

Output:

Growth cone extension

or

Growth cone collapse Current Opinion in Cell Biology

Ephrin/Eph signalling

Another major group of axon guidance molecules that utilise members of the Rho family of GTPases to regulate cell and growth cone motility is the Eph receptor family. Like other members of the receptor tyrosine kinase family, Eph receptors are transmembrane proteins with an extracellular ligand-binding domain and an intracellular cytoplasmic region having a tyrosine kinase domain [21,22]. The Eph receptors and their ligands, the ephrins, mediate cell-contact-dependent signalling and play important roles in the establishment of tissue patterns primarily by mediating repulsion [21,22]. Ephrin A ligands (ephrin A1–A5) are glycosylphosphatidylinositol-anchored molecules that bind EphA receptors (EphA1–A8) and play a critical role in axonal patterning in the nervous system, which includes the formation of the

retinotopic map in vertebrates [23–25]. In contrast to the A ephrins, the B ephrins (ephrin B1–B3) are transmembrane proteins with a cytoplasmic tail and bind to EphB (EphB1–B6) and EphA4 receptors [21,22]. The B ephrins and their receptors play important roles in axonal patterning in the midline, including the formation of the anterior commissure [26], sorting of retinal axons at the optic chiasm [27] and the crossing of corticospinal tract neurons in the spinal cord [28]. Whereas considerable progress has been made in understanding how ephrin/Eph expression profiles regulate patterning in the nervous system [21,22], the intracellular signalling mechanisms that transduce the signals into changes in growth cone motility are still poorly understood; however, a number of recent reports have begun to shed light on the molecular pathways involved in ephrin/Eph signalling.

224

Cell regulation

Figure 3

Figure 4 Semaphorin

Ephrin A1 EphA4 Plexin B

Ephexin

Rac-GTP

RhoA-GTP

Rac-GTP

Cdc42-GTP

RhoA-GTP

PAK1

PAK1 Growth cone collapse Current Opinion in Cell Biology

Model for Plexin-B-induced growth cone collapse. Plexin B inhibits Rac through a direct interaction of the Plexin B cytoplasmic tail with the GTPase. This reduces the ability of Rac to interact with and activate its downstream effector, PAK1. Plexin B also activates RhoA, also perhaps through a direct interaction. The combination of Rac inactivation and RhoA activation likely leads to growth cone collapse.

Ephexin

Recently, Greenberg and co-workers [29••] identified a GEF that interacts directly with the EphA4 receptor cytoplasmic domain. This protein called ephexin (for Eph-interacting exchange factor) acts as a direct link between Eph receptors and Rho GTPases. Ephexin is homologous to the Dbl family of GEFs and contains a unique aminoterminal region, a DH/PH domain (Dbl/plextrin homology region), which is required for binding to EphA4, and a Src homology 3 (SH3) domain. In the absence of stimulation, ephexin is constitutively bound to the EphA4 receptor cytoplasmic domain and is in a position to activate Cdc42 and Rac1 [29••]. In this state, growth cone extension is promoted. When EphA4 receptors are stimulated with a ligand (ephrin A1), ephexin activates RhoA while reducing the activation of Cdc42 and Rac1. The net effect is to induce growth cone collapse (Figure 4). Ligand binding is associated with the inhibition of PAK1, which is an effector of the Cdc42 and Rac GTPases [18,19,29••]. As PAK1 and its downstream effectors can remodel the actin cytoskeleton [18,19], a major function of ephexin may be to regulate cytoskeleton dynamics by modulating PAK1 activity. Further studies should help determine whether

Growth cone collapse Current Opinion in Cell Biology

Model for EphA4-induced growth cone collapse. When EphA4 receptors are stimulated with ephrin A1, ephexin activates RhoA while reducing the activation of Cdc42 and Rac1. Stimulation by ephrin A1 also leads to an inhibition of PAK1. The net effect is to induce growth cone collapse.

ephexin is required for signalling by the other EphA receptors, or possibly even the EphB receptors. PDZ–RGS3

Although much work has been performed on the signalling mechanisms downstream of the Eph receptors, the ephrin B ‘ligands’ have also been shown to be capable of signal transduction in what has been termed ‘reverse signalling’ [30–32]. Recently, Flanagan and co-workers [33••] have shown that reverse signalling is in part mediated by a novel protein that interacts with the cytoplasmic domain of ephrin B. This protein, PDZ–RGS3, contains a PDZ (PSD-95/Dlg/ZO-1) domain required for binding to ephrin B, and an RGS (regulator of G-protein signalling) domain, which interacts with heterotrimeric G proteins [34]. Proteins containing RGS domains have been shown previously to act as GAPs for the heterotrimeric G proteins that are associated with a large number of transmembrane receptors, including the chemokine receptor CXCR4 [35].

Axon guidance: the cytoplasmic tail Patel and Van Vactor

225

Figure 5 Model for ephrin B reverse signalling and potential crosstalk between PDZ–RGS3 and Grb4 signalling. (a) The binding of EphB to ephrin B activates PDZ–RGS3, which acts as a GAP towards heterotrimeric G proteins associated with G-protein-coupled receptors. This inhibits signalling downstream of the chemokine receptor CXCR4, thereby leading to a loss of chemotaxis towards its ligand, SDF-1. (b) Ephrin B activation can also lead to the recruitment of Grb4, an SH2/SH3 adaptor protein. Grb4 can interact with a number of different proteins that can remodel the actin cytoskeleton, including Abi-1, CAP and PAK1. The signalling pathways activated by Grb4 are likely to lead to actin depolymerisation and growth cone collapse. PDZ–RGS3 and Grb4 appear to bind distinct regions of the ephrin B cytoplasmic tail; however, it is not known if the effectors can bind simultaneously or if the binding of one effector might affect the binding of the other.

EphB

(a)

(b)

SDF-1

CXCR4

ephrin B

β γ

Gα GDP

-Y Grb4 PDZ-RGS3 ?

Gα GTP Abi-1

Inhibition of CXCR4 signalling and chemotaxis

CAP

PAK1

Actin depolymerisation Current Opinion in Cell Biology

These results suggest that, in vivo, reverse signalling by ephrin B may limit the outward migration of cerebellar granule neurons during development by inhibiting chemotaxis in response to SDF-1/CXCR4 signalling, and thus allow for the subsequent inward migration of these cells. It will be interesting to determine if the functions of ephrin B in retinal ganglion cell axon guidance [27,36] are also regulated by PDZ–RGS3 activation, and if other G-protein-coupled receptors, which may potentially play a role in axon guidance, can be targets of PDZ–RGS3.

important role in linking tyrosine kinases to intracellular signalling pathways, including those involving the Rho GTPases [38–40]. Grb4 contains an SH2 domain, which is required for binding to the cytoplasmic domain of ephrin B1, as well as three SH3 domains. Stimulation by soluble EphB2 leads to tyrosine phosphorylation of the ephrin B1 cytoplasmic domain, which in turn recruits Grb4 through its SH2 domain. Ephrin B1 activation results in a loss of polymerised actin in transfected cells and an increase in focal adhesion kinase (FAK) activity. The loss of actin polymerisation was inhibited by co-transfecting cells with a mutant Grb4 containing only the SH2 domain, which suggests that the SH3 domains are required for ephrin B1-mediated collapse. A yeast twohybrid screen and GST-pulldown assays identified a number of proteins that interact with the SH3 domains of Grb4. These included the Abl-interacting protein-1 (Abi-1), the Cbl-associated protein (CAP), and PAK1 (Figure 5b). All of these proteins have been implicated in cytoskeletal reorganisation [18,19,41,42], which suggests that Grb4 is likely to be a bona fide regulator of the cytoskeleton and axon guidance. The large number of proteins that Grb4 interacts with also suggests the possibility of a great deal of crosstalk between various signalling pathways.

Grb4

Robo

Cowan and Henkemeyer [37••] recently found that the adapter protein Grb4 also transduces ephrin B reverse signals. Grb4 is an adapter protein closely related to, but distinct from, Nck/DOCK, which has been shown to play an

Another receptor that has been shown recently to regulate the Rho family of GTPases is Robo, which plays a major role in axon guidance at the midline of the nervous system. In the midline of vertebrates, insects and nematodes,

In an elegant series of experiments, the researchers demonstrate that the binding of EphB to ephrin B activates the RGS domain of PDZ–RGS3 and that this activation inhibits signalling downstream of CXCR4. SDF-1, the ligand for CXCR4, is a chemoattractant for cerebellar granule cells and promotes their outward migration towards the pial membrane [35]. In vitro, SDF-1 was able to promote the chemotaxis of cerebellar granule cells; however, simultaneous activation of ephrin B by the addition of soluble EphB inhibited migration in response to SDF-1, which suggests that PDZ–RGS3 inhibits CXCR4 signalling ([33]; Figure 5a).

226

Cell regulation

Figure 6

Netrin

Slit

Robo

(a)

DCC

P _ CC1

Abl

P2

*

P3

CC2

srGAP

(b)

P1

CC0

Model of a dual role for Robo in guidance. (a) Mammalian Robo1 transduces part of the signal for repulsion by directly interacting with srGAPs. The binding of Slit to Robo1 recruits srGAP to the receptor’s cytoplasmic tail. This is associated with the activation of RhoA and the inhibition of Cdc42. This shift in the balance of GTPase activation may lead to growth cone collapse and repulsion. (b) Robo1 can also silence signalling by the netrin receptor DCC in a direct form of crosstalk between the two receptors. The binding of Slit to Robo1 induces an interaction between the cytoplasmic domains of Robo1 and DCC (*). This involves the CC1 domain in Robo1 and the P3 domain in DCC. This direct interaction prevents DCC from transmitting the signal for attraction upon netrin binding. At present, it is not known if Robo1 can transduce the srGAP-mediated signal for repulsion and silence attraction by DCC simultaneously. In addition, it is not known how phosphorylation of the Robo CC1 domain by the tyrosine kinase Abl (dashed arrow) may affect signalling by the srGAP pathway or the ability of Robo to silence attraction by DCC.

CC3

X

*Slit-dependent silencing

Attraction Cdc42-GTP

RhoA-GTP

Repulsion Current Opinion in Cell Biology

commissural axons are attracted by netrin proteins secreted by midline cells that activate a receptor of the DCC (deleted in colorectal cancer) family on growth cones [43]. After crossing the midline, the axons cease to be attracted to the midline and are instead repelled from it; thus, ensuring that they do not recross. The repulsion is mediated, in part, by an increased sensitivity to repellents on the portion of the axon that has crossed the midline so that the axon is now responsive to repellents such as Slit and semaphorin [44–48]. Axons that cross the midline also lose their responsiveness to the netrin attractant, although they still continue to express the DCC receptor [48]. In Drosophila and vertebrates, the Robo family of Slit receptors mediates repulsion and plays an important role in the decision of axons to cross the midline [48–53]. In robo mutant Drosophila embryos, too many axons cross the midline where the ligand for the Robo receptors, Slit, is produced by glial cells [46–49,53].

A report from Wong et al. [54••] now demonstrates that mammalian Robo1 transduces part of the signal for repulsion, by directly interacting with a novel family of GAPs, the Slit–Robo GAPs (srGAPs). In transfected cells, the binding of Slit to Robo1 was found to increase the interaction of srGAP1 with the receptor’s cytoplasmic domain and was accompanied by the activation of RhoA and the inhibition of Cdc42. The fact that, in vitro, srGAP1 associates with Cdc42 suggests that srGAP1 directly modulates Cdc42 activity. Furthermore, dominantnegative srGAP1 blocked Slit-mediated inactivation of Cdc42 and repulsion in cell culture. A constitutively active Cdc42 blocked the repulsive effect of Slit ([54••]; Figure 6a); therefore, these results reveal the importance of Cdc42 in Robo signalling and indicate that srGAP1 mediates repulsion by inhibiting Cdc42. Future experiments, including genetic experiments in Drosophila, should reveal if Cdc42 and the other Rho-family

Axon guidance: the cytoplasmic tail Patel and Van Vactor

GTPases participate in axon guidance downstream of Robo in vivo. Whereas the Rho GTPases play a role in mediating Robo’s output, a recent report from Stein and Tessier-Lavigne [55••] demonstrates that Robo can silence attraction to the midline as well, in a direct form of crosstalk with the netrin receptor DCC. These investigators found that the Robo cytoplasmic domain can inhibit signalling by DCC [55••]. In transfected Xenopus neurons, the binding of Slit to Robo induces a direct interaction between the cytoplasmic domains of Robo and DCC. This interaction prevents DCC from transmitting the signal for attraction upon netrin binding. The interaction of the cytoplasmic tails of the two receptors is mediated by short conserved domains in each receptor — CC1 in Robo and P3 in DCC (Figure 6b). Stein and Tessier-Lavigne [55••] were able, with a pair of chimeric receptors, to mimic the physical association and the silencing effect. These receptors were activated by two completely different ligands and associated through different interaction domains, providing further evidence that the silencing occurs at the level of the cytoplasmic domains [55••]. These results suggest that the activation of Robo by Slit not only serves to repel axons from the midline, but also switches off attraction to it; thus, ensuring that they do not recross (Figure 6). It is not yet known if Robo-mediated silencing of netrin attraction occurs in vivo; however, the Robo family of receptors in mammals and Drosophila are highly conserved, with all three members in Drosophila (Robo1–3) containing the conserved CC1 domain [49–52,55••]. In addition, the Drosophila netrin receptor Frazzled is highly homologous to DCC and also contains the cytoplasmic P3 domain [56]; thus, this provides the opportunity to perform genetic studies in Drosophila and to examine if Robo-mediated silencing of attraction to the midline occurs in vivo.

The cytoplasmic domain in signalling and crosstalk These and other studies (e.g. [57,58•]) highlight the importance of the cytoplasmic domain in transmitting guidance cues. They also emphasise that the cytoplasmic domains are the sites where signals are not only transmitted but are regulated as well. As an increasing number of cytoplasmic signalling partners are identified for each class of axon guidance receptor, the next wave of questions will address the interactions that occur between the various signalling pathways. This will help clarify how receptor activation leads to an integrated output that translates the multiple axon guidance cues at the cell surface into coordinated changes in growth cone motility. Future work should shed light on the regulation of receptor signalling and the cross talk that occurs between the various signalling pathways. For example, it is important to examine in which contexts Grb4 or PDZ–RGS3 signalling predominates during ephrin B

227

reverse signalling. Although Grb4 and PDZ–RGS3 appear to bind distinct regions of the ephrin B cytoplasmic domain [33••,37••], it is not known if the binding of the two effectors is mutually exclusive or if both can bind the receptor simultaneously (Figure 5). Furthermore, these interactions are likely to be regulated by EphB binding, because phosphorylation of the ephrin B cytoplasmic domain, which occurs upon binding to EphB, recruits Grb4 through its SH2 domain. In addition, it will be worth investigating if PDZ–RGS3 can regulate other heterotrimeric G proteins that may play a role in axon guidance [59,60]. Future work should also clarify how Robo signalling is modulated and the crosstalk that occurs with other signalling pathways. In addition to regulating the Rho GTPases, Robo has been shown to directly interact with the non-receptor tyrosine kinase Abelson (Abl) as well as its substrate Enabled (Ena), which is involved in mediating Robo signalling, and with Abl antagonising Robo’s output [58•]. The ability of Abl to inhibit the repulsive output of Robo is likely to be due to its ability to phosphorylate the cytoplasmic tail of the receptor. Indeed a mutant Robo lacking an Abl-phosphorylation site in the CC1 domain leads to a hyperactive receptor [58•]. Future experiments should address if the phosphorylation of the Robo cytoplasmic domain by Abl affects the interaction of srGAP with Robo, and its ability to modulate GTPase activity and mediate repulsion. Additionally, Abl-mediated phosphorylation might also affect the ability of Robo to silence attraction by the netrin receptor, as silencing requires the CC1 domain in Robo; thus, it will be worthwhile examining how phosphorylation affects Robo’s repulsive output and its ability to silence attraction at the midline (Figure 6). Intriguingly, the overall plan in Robo signalling is very similar to that of the receptor tyrosine phosphatase DLAR, which plays an important role in photoreceptor and motor axon guidance in Drosophila [61–64]. Akin to the Robo pathway, Ena is required for DLAR’s output, whereas Abl is an antagonist of the receptor [62]. Furthermore, DLAR also genetically interacts with a GTPase, Rac1 and the GEF Trio [63–66]. Although it is not known if Robo interacts with either Rac1 or Trio, the similarity between the Robo and DLAR signalling pathways suggests that a convergence between Abl and GTPase pathways may be a theme common to numerous guidance receptors. It will be very interesting to see whether this translates into crosstalk between the Robo and phosphatase signalling pathways.

Conclusions Considerable work is required to decipher the elaborate signalling mechanisms used by the various receptors to finely regulate axon guidance. Nonetheless, a combination of biochemical, cell culture and in vivo experiments, such as that described here, should contribute to a better understanding of the signalling mechanisms that regulate axon guidance. In addition to the GTPases, many other effectors are likely to be found downstream of the

228

Cell regulation

guidance receptors, such as a multitude of non-receptor tyrosine and serine/threonine kinases and proteins that effect the growth cone cytoskeleton [67]. A great deal of crosstalk between the different receptor signalling pathways is also likely, which has been emphasised by the recent work. Future work should reveal further examples of crosstalk, at the level of receptor-cytoplasmic domains as well as at the level of downstream mediators. Ultimately, a better understanding of the intricate receptor signalling mechanisms will help reveal how a multiplicity of cues at the cell surface are translated into the stereotyped changes in growth cone responsiveness and motility that are necessary during the development of the nervous system.

Acknowledgements We would like to thank Mark Emerson, Karl Johnson and Jannette Rusch for critical discussion. BN Patel is supported by a fellowship from the Canadian Institutes of Health Research. D Van Vactor is a Leukemia and Lymphoma Society fellow. DVV and BNP are also supported by National Institutes of Health (NIH) grants NS40043 and NS35909.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest

16. Vikis HG, Li W, He Z, Guan KL: The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proc Natl Acad Sci USA 2000, 97:12457-12462. 17. •

Hailan Hu, Marton TF, Goodman CS: Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron 2001, 32:39-51. This study demonstrates that the semaphorin receptor Plexin B inhibits Rac through a direct interaction of the receptor’s cytoplasmic domain with the GTPase. Plexin B was also found to activate RhoA. The authors suggest that the combined inhibition of Rac and activation of RhoA leads to growth cone collapse. 18. Edwards DC, Sanders LC, Bokoch GM, Gill GN: Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1999, 1:253-259. 19. Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P: Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999, 283:2083-2085. 20. Winberg ML, Noordermeer JN, Tamagnone L, Comoglio PM, Spriggs MK, Tessier-Lavigne M, Goodman CS: Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell 1998, 95:903-916. 21. Schmucker D, Zipursky SL: Signaling downstream of Eph receptors and ephrin ligands. Cell 2001, 105:701-704. 22. Wilkinson DG: Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci 2001, 2:155-164. 23. Feldheim DA, Vanderhaeghen P, Hansen MJ, Frisen J, Lu Q, Barbacid M, Flanagan JG: Topographic guidance labels in a sensory projection to the forebrain. Neuron 1998, 21:1303-1313. 24. Brown A, Yates PA, Burrola P, Ortuno D, Vaidya A, Jessell TM, Pfaff SL, O’Leary DD, Lemke G: Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling. Cell 2000, 102:77-88.

1.

Mueller BK: Growth cone guidance: first steps towards a deeper understanding. Annu Rev Neurosci 1999, 22:351-388.

2.

Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell 2000, 103:211-225.

3.

Simon MA: Receptor tyrosine kinases: specific outcomes from general signals. Cell 2000, 103:13-15.

26. Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder J, Pawson T, Klein R: Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 1996, 86:35-46.

4.

Stoker AW: Receptor tyrosine phosphatases in axon growth and guidance. Curr Opin Neurobiol 2001, 11:95-102.

27.

5.

Kamiguchi H, Lemmon V: IgCAMs: bidirectional signals underlying neurite growth. Curr Opin Cell Biol 2000, 12:598-605.

6.

Ranscht B: Cadherins: molecular codes for axon guidance and synapse formation. Int J Dev Neurosci 2000, 18:643-651.

7.

Tamagnone L, Comoglio PM: Signalling by semaphorin receptors: cell guidance and beyond. Trends Cell Biol 2000, 10:377-383.

8.

Raper JA: Semaphorins and their receptors in vertebrates and invertebrates. Curr Opin Neurobiol 2000, 10:88-94.

9.

Kennedy TE: Cellular mechanisms of netrin function: long-range and short-range actions. Biochem Cell Biol 2000, 78:569-575.

10. Giger RJ, Kolodkin AL: Silencing the siren: guidance cue hierarchies at the CNS midline. Cell 2001, 105:1-4. 11. Dickson BJ: Rho GTPases in growth cone guidance. Curr Opin Neurobiol 2001, 11:103-110. 12. Kozma R, Sarner S, Ahmed S, Lim L: Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol Cell Biol 1997, 17:1201-1211. 13. Liu BP, Strittmatter SM: Semaphorin-mediated axonal guidance via Rho-related G proteins. Curr Opin Cell Biol 2001, 13:619-626. 14. Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H, Chedotal A, Winberg ML, Goodman CS, Poo M et al.: Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 1999, 99:71-80. 15. Driessens MH, Hu H, Nobes CD, Self A, Jordens I, Goodman CS, Hall A: Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr Biol 2001, 11:339-344.

25. Feldheim DA, Kim YI, Bergemann AD, Frisen J, Barbacid M, Flanagan JG: Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron 2000, 25:563-574.

Nakagawa S, Brennan C, Johnson KG, Shewan D, Harris WA, Holt CE: Ephrin-B regulates the ipsilateral routing of retinal axons at the optic chiasm. Neuron 2000, 25:599-610.

28. Yokoyama N, Romero MI, Cowan CA, Galvan P, Helmbacher F, Charnay P, Parada LF, Henkemeyer M: Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline. Neuron 2001, 29:85-97. 29. Shamah SM, Lin MZ, Goldberg JL, Estrach S, Sahin M, Hu L, •• Bazalakova M, Neve RL, Corfas G, Greenberg ME et al.: EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin. Cell 2001, 105:233-244. The authors identify a novel guanine nucleotide exchange factor, ephexin, which interacts directly with the EphA4 receptor cytoplasmic domain. When EphA4 receptors are stimulated with ephrin A1, ephexin activates RhoA while reducing the activation of Cdc42 and Rac1. This shift in the balance of GTPase activation is thought to lead to growth cone collapse. 30. Holland SJ, Gale NW, Mbamalu G, Yancopoulos GD, Henkemeyer M, Pawson T: Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands. Nature 1996, 383:722-725. 31. Bruckner K, Pasquale EB, Klein R: Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science 1997, 275:1640-1643. 32. Bruckner K, Pablo Labrador J, Scheiffele P, Herb A, Seeburg PH, Klein R: EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron 1999, 22:511-524. 33. Lu Q, Sun EE, Klein RS, Flanagan JG: Ephrin-B reverse signaling is •• mediated by a novel PDZ-RGS protein and selectively inhibits G protein-coupled chemoattraction. Cell 2001, 105:69-79. The authors identify a novel protein, PDZ–RGS, which associates with the ephrin B cytoplasmic domain and is activated upon EphB binding. This leads to the inhibition of signalling through the G-protein-coupled receptor CXCR4, and the loss of chemotaxis towards its ligand SDF-1.

Axon guidance: the cytoplasmic tail Patel and Van Vactor

34. Ross EM, Wilkie TM: GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 2000, 69:795-827. 35. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR: Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998, 393:595-599. 36. Birgbauer E, Oster SF, Severin CG, Sretavan DW: Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues. Development 2001, 128:3041-3048. 37. Cowan CA, Henkemeyer M: The SH2/SH3 adaptor Grb4 •• transduces B-ephrin reverse signals. Nature 2001, 413:174-179. The authors demonstrate that the adaptor protein Grb4 is recruited to the ephrin B1 cytoplasmic domain upon EphB2 binding. The loss of actin polymerisation that accompanies EphB2 binding can be inhibited using a mutant Grb4, which suggests that it is required for ephrin-B1-mediated collapse. Grb4 was also found to interact with proteins known to remodel the cytoskeleton, including Abi-1, CAP and PAK1. 38. Garrity PA, Rao Y, Salecker I, McGlade J, Pawson T, Zipursky SL: Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein. Cell 1996, 85:639-650. 39. Braverman LE, Quilliam LA: Identification of Grb4/Nckbeta, a Src homology 2 and 3 domain-containing adapter protein having similar binding and biological properties to Nck. J Biol Chem 1999, 274:5542-5549. 40. Newsome TP, Schmidt S, Dietzl G, Keleman K, Asling B, Debant A, Dickson BJ: Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell 2000, 101:283-294. 41. Stradal T, Courtney KD, Rottner K, Hahne P, Small JV, Pendergast AM: The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol 2001, 11:891-895. 42. Thien CB, Langdon WY: Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol 2001, 2:294-307. 43. Kennedy TE: Cellular mechanisms of netrin function: long-range and short-range actions. Biochem Cell Biol 2000, 78:569-575.

229

53. Tear G: A new code for axons. Nature 2001, 409:472-473. 54. Wong K, Ren X-R, Huang Y-Z, X Yi, Liu G, Saito H, Tang H, Wen L, •• Brady-Kalnay SM, Mei L et al.: Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit–Robo pathway. Cell 2001, 107:209-221. The authors identify a novel family of GTPase-activating proteins (GAPs) — the Slit–Robo GAPs (srGAPs) — that interact with the Robo cytoplasmic domain and transduce the signal for repulsion. The binding of Slit to Robo1 increases the interaction of srGAP1 with the receptor’s cytoplasmic domain. This is associated with the inhibition of Cdc42 and the activation of RhoA. Dominant-negative srGAP1 was found to block Slit-mediated repulsion in vitro, arguing that srGAP1 is required for Robo’s repulsive output. 55. Stein E, Tessier-Lavigne M: Hierarchical organization of guidance •• receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 2001, 291:1928-1938. The authors demonstrate that Robo can silence netrin-mediated attraction. The activation of Robo by Slit binding leads to a direct interaction of the cytoplasmic domains of Robo and the netrin receptor DCC (deleted in colorectal cancer). This interaction prevents DCC from transmitting the signal for attraction. 56. Kolodziej PA, Timpe LC, Mitchell KJ, Fried, SR, Goodman CS, Jan LY, Jan YN: Frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 1996, 87:197–204. 57.

Bashaw GJ, Goodman CS: Chimeric axon guidance receptors: The cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion. Cell 1999, 97:917-926.

58. Bashaw GJ, Kidd T, Murray D, Pawson T, Goodman CS: Repulsive • axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 2000, 101:703-715. The authors show that the tyrosine kinase Abelson (Abl) and its substrate Enabled (Ena) act downstream of Robo. Ena is found to be part of Robo’s output, whereas Abl antagonises Robo signalling. In vitro, Abl phosphorylates the Robo CC1 domain; and a Robo receptor lacking the tyrosine phosphorylation site is hyperactive, which suggests that Abl-mediated phosphorylation of Robo negatively regulates Robo signalling.

44. Kidd T, Russell C, Goodman CS, Tear G: Dosage-sensitive and complementary functions of roundabout and commissureless control axon crossing of the CNS midline. Neuron 1998, 20:25-33.

59. Katoh H, Aoki J, Yamaguchi Y, Kitano Y, Ichikawa A, Negishi M: Constitutively active Galpha12, Galpha13, and Galphaq induce Rho-dependent neurite retraction through different signaling pathways. J Biol Chem 1998, 273:28700-28707.

45. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, Tear G: Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 1998, 92:205-215.

60. Nakayama T, Goshima Y, Misu Y, Kato T: Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse. J Neurobiol 1999, 41:326-339.

46. Kidd T, Bland KS, Goodman CS: Slit is the midline repellent for the Robo receptor in Drosophila. Cell 1999, 96:785-794. 47.

Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T: Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 1999, 96:795-806.

48. Zou Y, Stoeckli E, Chen H, Tessier-Lavigne M: Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 2000, 102:363-375.

61. Krueger NX, Van Vactor D, Wan HI, Gelbart WM, Goodman CS, Saito H: The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 1996, 84:611-622. 62. Wills Z, Bateman J, Korey CA, Comer A, Van Vactor D: The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 1999, 22:301-312. 63. Kaufmann N, Wills ZP, Van Vactor D: Drosophila Rac1 controls motor axon guidance. Development 1998, 125:453-461.

49. Rajagopalan S, Nicolas E, Vivancos V, Berger J, Dickson BJ: Crossing the midline: roles and regulation of Robo receptors. Neuron 2000, 28:767-777.

64. Maurel-Zaffran C, Suzuki T, Gahmon G, Treisman JE, Dickson BJ: Cell-autonomous and -nonautonomous functions of LAR in R7 photoreceptor axon targeting. Neuron 2001, 32:225-235.

50. Rajagopalan S, Vivancos V, Nicolas E, Dickson BJ: Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 2000, 103:1033-1045.

65. Bateman J, Shu H, Van Vactor D: The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo. Neuron 2000, 26:93-106.

51. Simpson JH, Bland KS, Fetter RD, Goodman CS: Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position. Cell 2000, 103:1019-1032.

66. Liebl EC, Forsthoefel DJ, Franco LS, Sample SH, Hess JE, Cowger JA, Chandler MP, Shupert AM, Seeger MA: Dosage-sensitive, reciprocal genetic interactions between the Abl tyrosine kinase and the putative GEF trio reveal trio’s role in axon pathfinding. Neuron 2000, 26:107-118.

52. Simpson JH, Kidd T, Bland KS, Goodman CS: Short-range and longrange guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance. Neuron 2000, 28:753-766.

67.

Song H, Poo M: The cell biology of neuronal navigation. Nat Cell Biol 2001, 3:E81-E88.