CAMs and Axonal Growth: A Critical Evaluation of the Role of Calcium and the MAPK Cascade

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Molecular and Cellular Neuroscience 16, 283–295 (2000)

doi:10.1006/mcne.2000.0907, available online at http://www.idealibrary.com on MCN

A

REVIEW

CAMs and Axonal Growth: A Critical Evaluationof the Role of Calcium and the MAPK Cascade

Patrick Doherty,1 Gareth Williams, and Emma-Jane WilliamsMolecular Neurobiology Group, MRC Centre for Developmental Neurobiology,King’s College London, London Bridge, London SE1 1UL, United Kingdom

Calcium has long been recognized as a key player in thecontrol of axonal growth and guidance. Recent studieslend support to this pivotal role by showing that localchanges in calcium can directly induce the formation offilopodia in vivo and turn a growth cone in vitro. Under

ormal growth conditions, the L1 adhesion molecule hasow been shown to induce local rather than globalhanges in calcium in growth cones, and this suggestshat cell adhesion molecules (CAMs) use localized cal-ium transients to stimulate axonal growth and guidance.number of recent reports have demonstrated that the

eurite outgrowth response stimulated by L1 and otherdhesion molecules (NCAM, N-cadherin, laminin) also de-ends in part upon the integrity of the MAPK cascade inells. In this review we consider the recent data and sug-est that calcium and the MAPK cascade might be re-uired for very distinct growth cone functions. Finally, weill consider the contentious issue of how the aboveAMs activate signaling cascades in growth cones and

eview the recently available data that support the hypoth-sis that at least one of these CAMs (N-cadherin) mightromote growth cone motility by directly interacting withhe FGFR in growth cones.

INTRODUCTION

The growth cone at the tip of an extending axonsamples its local environment and makes decisions onwhere to navigate to in the developing embryo. Animportant role for various members of the integrin,cadherin, and Ig superfamilies of adhesion molecules in

1 To whom correspondence and reprint requests should be ad-ressed. Fax: 0207 848 6813. E-mail: [email protected].

1044-7431/00 $35.00Copyright © 2000 by Academic Press

ll rights of reproduction in any form reserved.

the process of axonal growth and guidance has nowbeen established (reviewed in Bixby and Harris, 1991;Reichardt and Tomaselli, 1991; Tessier-Lavigne andGoodman, 1996; Walsh et al., 1997). The full range anddiversity of the CAMs that are candidates for involve-ment in this process is only now starting to becomeapparent. In this context, Hynes and Zhao have identi-fied 500 genes (;4% of the genome) from the Drosoph-ila genome project that might be directly involved inadhesion (Hynes and Zhao, 2000). A significant subsetwill be expressed in the developing brain and will havethe potential to modulate growth cone motility. Itshould be borne in mind that additional molecular andfunctional diversity can arise from alternative splicingand differential posttranslational processing of individ-ual gene products.

A range of experimental paradigms have been usedto demonstrate that the function of a large number ofdistinct classes of molecule including Ig CAMs can playa role in individual developmental events, such as ax-onal guidance at midline structures (reviewed in Sto-eckli and Landmesser, 1998; Tear, 1999). Thus, a fullunderstanding of the molecular basis of neural devel-opment will require an understanding not only of howa single molecule works, but also of how a growth coneintegrates information gleaned from a number of path-way cues. In this context, although it is now widelyaccepted that CAMs can promote axonal growth byactivating signal transduction cascades in growth cones(reviewed in Walsh and Doherty, 1997), we are stilllacking in a detailed understanding of how any singleCAM actually does this.

In this review we will focus our attention on NCAM,N-cadherin, and L1. This is for three reasons. First,

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284 Doherty, Williams, and Williams

these CAMs were amongst the first to be implicated inthe control of axonal growth, guidance and regenera-tion (reviewed in Doherty and Walsh, 1989). Second,the signaling potential of these CAMs in neurons wasdemonstrated as long ago as 1989 (Schuch et al., 1989)and shown to be important for axonal growth in theearly 1990s (Doherty et al., 1991). Finally, recent obser-vations in man have shown clear defects in axonalpathways when the L1 adhesion molecule is not ex-pressed during development, or a mutant form is ex-pressed (reviewed in Kenwrick et al., 2000). Our narrowfocus should not detract from the important role thatother molecular families play in the process of axonalgrowth and guidance including the semaphorins andtheir receptors (Goodman, 1996) and the ephrins andtheir ligands (Wilkinson, 2000). However, lessonslearned about the signaling cascades activated byNCAM, N-cadherin, and L1 will probably yield insightsinto the nature of the pathways that mediate responsesfrom other guidance cues given that they all impinge ongrowth cone motility.

THERE IS MORE TO CAM FUNCTIONTHAN ADHESION

NCAM, N-cadherin, and L1 were established as ad-hesion molecules (Hatta et al., 1985; Rathjen andSchachner, 1984; Rutishauser et al., 1982; Sadoul et al.,983) prior to the observation that they could also di-ectly promote axonal growth in vitro via a homophilicinding mechanism (Doherty et al., 1990; Lemmon et al.,989; Matsunaga et al., 1988). Given the fact that differ-ntial adhesion can modulate growth cone guidanceLetourneau, 1975), it seemed reasonable to assume thathe above CAMs were promoting axonal growth byirectly modulating the adhesion of the growth cone toellular substrates. However, a series of papers fromur own and other labs throughout the 1990s has nowstablished that this is not the case. Most workers nowgree that the ability of these CAMs to activate intra-ellular signaling cascades is fundamental to their abil-ty to promote axonal growth and guidance. This iserhaps best illustrated by the fact that soluble versionsf L1 (Doherty et al., 1995b), NCAM (Meiri et al., 1998),nd N-cadherin (Doherty lab, unpublished observation)an all promote neurite outgrowth as effectively as theame CAM expressed on the surface of a transfected
ell. The ability of CAMs to support cell adhesion and
their ability to activate signaling cascades most likelygm

accounts for the fact that at some developmental stagesa CAM might actively promote cell migration, axonalgrowth and synaptic plasticity, whereas at other devel-opmental stages the same CAM might serve to stabilizecell connections. In this context, studies on the neuralcell adhesion molecule (NCAM) have shown that use ofa small exon called the VASE exon, and/or loss ofpolysialic acid, can dramatically change the function ofNCAM from a molecule that promotes neurite out-growth and synaptic plasticity to one that probablyfunctions to maintain synaptic structures (Doherty et al.,1995a).

TWO COMPARTMENTS: A SIMPLEMODEL OF A GROWTH CONE

The growth cone is a highly dynamic and specializedstructure adapted for axonal growth and guidance (re-viewed in Gordon-Weeks, 2000) (Fig. 1a). By interactingwith cues in the local environment, receptor moleculeson the lamellipodia and/or filopodia trigger a cascadethat ultimately leads to the assembly and/or disassem-bly of force-generating machinery within the leadingedge of the growth cone. Growth cone movement alsodepends on the transport of membrane bound vesiclesfrom the cell body to the growth cone and possibly alsothe recycling of membrane vesicles via the process ofendocytosis and membrane fusion within the growthcone itself (Figs. 1b and 1c). For appropriate growthcone motility, all of the components (for example, re-ceptors, adapters, kinases, phosphatases, cytoskeletalelements, vesicular transport pathways) have to presentand function in a coordinated manner. There will alsohave to be mechanisms in place to consolidate the earlyconsequences of the signaling, mechanisms to turn offsignals at the “old leading edge” as the growth coneadvances, and mechanisms to then recycle parts of thesignaling machinery back to the new leading edge.Even in this highly simplified model, we can envisagethat signals generated within the leading edge (P do-main) and the organelle rich body of the growth cone (Cdomain) most likely have distinct functions in control-ling growth cone motility. Given the dynamics ofgrowth cone motility (see Gordon-Weeks, 2000), itseems reasonable to characterize signals generated atthe P domain as being instructive signals for growth and

uidance, whereas those generated at the C domain areore likely to be permissive signals.

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285CAMs and Axonal Growth

LOCALIZED CALCIUM CAN STEERTHE GROWTH CONE

Calcium is a key second messenger within growthcones that can both increase the rate of growth coneextension, turn growth cones and induce growth conecollapse (Gundersen and Barrett, 1980; Mattson andKater, 1987; McCaig, 1989; Silver et al., 1989; Snow et al.,990, 1994; Suarez-Isla et al., 1984; Williams et al., 1992).he differential effects of calcium on growth cone mo-

ility (e.g., growth and retraction) can be explained in

FIG. 1. A simple schematic of the growth cone that illustrates theegion (the C-domain) and the peripheral microfilament rich region (tn red. In (b) and (c) we depict membrane bound vesicles arriving inan be transported from the cell body along microtubules, or generas controlled by dynamic changes in the cytoskeleton, however, grow

embrane into the growth cone (reviewed in Gordon-Weeks, 2000).IG. 2. A schematic diagram showing an FGFR-activated signaling cCAM, N-cadherin, L1, and FGF. The initial step involves recruitmen

will then stimulate the production of diacylglycerol (DAG), whichArachidonic acid stimulates a very localized increase in calcium concchannel antagonists. GAP43 is then phosphorylated by a membranePhosphorylated GAP43 no longer binds calmodulin, and the freeCa21/calmodulin kinase (Cam Kinase). In support of the above schenhibit PLCg, DAG lipase, calcium influx into cells and Ca21/calmo

response to CAMs and FGF. (3) CAMs and FGF activate this cascadsynthesis, and in growth cones as determined by calcium/barium imneurons, activation of this cascade at the level of arachidonic acid pCAM and FGF (see text for details).

art by the “set-point messenger” hypothesis (Katernd Mills, 1991). In this scheme, there is an optimal or

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et-point level of calcium that promotes maximalrowth. Increasing calcium levels toward the set-pointill increases motility, whereas increasing it above the

et-point decreases motility. Although this schemeolds well in some circumstances (e.g., see al-Mohannat al., 1992) in other circumstances calcium influx intoeurons can stimulate neurite outgrowth even though

he basal level of calcium is above the set-point levelHarper et al., 1994). It is now recognized that bothpatial and temporal changes in calcium concentrationre likely to play determining roles in growth cone

ain compartments. In (a) we illustrate the central microtubule richdomain) by drawing the microtubules in blue and the actin filamentsrowth cone and ultimately fusing at the leading edge. Such vesiclesithin the growth cone by endocytosis. Axonal growth and guidanceo depends upon vesicle trafficking and a continued insertion of new

e that can account for the neurite outgrowth response stimulated byPLCg to the activated FGFR (not shown) at the cell membrane. PLCgubsequently hydrolyzed to arachidonic acid (AA) by DAG lipase.tion via channels that can be inhibited by N- and/or L-type calciumnd isoform of PKC that is activated by AA and/or calcium influx.odulin will be available to act with the calcium to activate the

1) CAM and FGF responses are inhibited by agents that specificallyn kinase activity. (2) Neurons that lack GAP43 show a diminishedneurons as determined by direct measurement of arachidonic acid

g and/or the direct assay of GAP43 phosphorylation. (4) in primarytion can fully mimic the neurite outgrowth response stimulated by

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Given their low cytoplasmic volume lamellipodia andfilopodia will be particularly sensitive to small move-ments of calcium and this has been formally demon-strated for filopodia (Davenport et al., 1993; Davenportand Kater, 1992). Although filopodia are not requiredfor axonal growth per se, they are required for appro-priate guidance (Bentley and Toroian-Raymond, 1986)and the behavior of this relatively simple structure canpredict and determine the behavior of the entire growthcone (O’Connor et al., 1990; Zheng et al., 1996). There-fore, calcium signals that control axonal growth andguidance are expected to be generated within filopodiaand/or lamellipodia at the leading edge of the growthcone. Recent studies using caged calcium compoundshave demonstrated that localized changes in calcium ingrowth cones can induce the formation of new filopodiain vivo (Lau et al., 1999), and also induce growth coneturning responses in vitro (Zheng, 2000). It can therefore

e concluded that localized changes in calcium canromote both axonal growth and guidance in an in-

structive manner.

CAMS INDUCE LOCALIZED CHANGESIN CALCIUM IN GROWTH CONES

Calcium influx into neurons through both N- andL-type calcium channels is required for, and is sufficientto drive, the neurite outgrowth responses stimulated byNCAM, N-cadherin, and L1 (Doherty et al., 1991; Walshand Doherty, 1997; Williams et al., 1992). However,there is no detectable increase in steady state levels ofcalcium in the neuron when neurons are actively grow-ing in response to L1 (Harper et al., 1994). Likewise,although a soluble L1-Fc chimera can stimulate calci-um-dependent neurite outgrowth (Archer et al., 1999;Doherty et al., 1995b) it has no measurable effect oncalcium concentration in the body of the growth cone(Archer et al., 1999). We postulated that this paradoxmight be resolved by the process of “calcium cycling.”In this scheme calcium influx is balanced by the rapidextrusion of calcium by membrane pumps. Any in-crease in calcium concentration would be restricted tothe site of calcium entry, and we would not expectconventional calcium imaging to detect such a change.Barium is a poor substrate for the calcium extrusionpumps, however, it can readily pass through open cal-cium channels. Using barium as the charge carrier, the
Bolsover lab has recently provided the first direct evi-dence that CAM stimulated neurite outgrowth is driven
by localized submembrane increases in calcium concen-tration in growth cones, rather than global changes(Archer et al., 1999). The importance of the local natureof the calcium signal should not be underestimated asthis limits the action of calcium to effector moleculesthat are localized near to the membrane (for examplePKCbII and GAP-43, see below). Because the calciumdoes not diffuse far, it provides a mechanism for local-izing a signal to a distinct region of a growth cone andthis is important for growth cone turning.

THE FGFR COUPLES CAMSTO CALCIUM INFLUX

Neurite outgrowth stimulated by NCAM, N-cad-herin, and L1 is dependent on the tyrosine kinase ac-tivity of the FGFR in neurons (Kolkova et al., 2000; Lomet al., 1998; Ronn et al., 2000; Saffell et al., 1997; Williamset al., 1994a). Much of this work depended on the use ofa kinase deleted dominant negative form of the FGFR,but it is also supported by the fact that antibodies to theFGFR, and agents that inhibit the FGFR–PLCg receptorsignaling cascade (see below), also inhibit the CAMresponses (reviewed in Walsh and Doherty, 1997). Insome circumstances expression of a dominant negativeFGFR can inhibit NGF responses by sequestering FRS2by a mechanism that does not require an active kinase(Ong et al., 2000). However, in the CAM studies citedabove the dominant negative FGFR did not inhibit neu-rite outgrowth stimulated by integrins, or activation ofTrk receptors by NGF or BDNF (Lom et al., 1998; Ronnet al., 2000; Saffell et al., 1997). Furthermore, we haverecently found that a highly specific FGFR antagonist(PD 173074) (Mohammadi et al., 1998) can specificallyinhibit the neurite outgrowth response stimulated by allthree CAMs (E.W., unpublished observation).

Soluble chimeric forms of L1 and NCAM can alsopromote neurite outgrowth in an FGFR-dependentmanner (Doherty et al., 1995b; Meiri et al., 1998). TheseCAMs can also activate the FGFR in PC12 cells (Saffellet al., 1997) and activation of the FGFR with FGF2mimics the neurite outgrowth response stimulated bythe above three CAMs (Williams et al., 1994a,b). Therecent studies from the Bolsover lab have shown thatactivation of the FGFR by FGF2 also triggers localizedchanges, rather than global changes, in calcium ingrowth cones (Archer et al., 1999). The CAM stimulated
calcium influx into growth cones was also shown to bedependent on the kinase activity of the FGFR. Based on

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these results we can conclude that CAMs stimulateaxonal growth by activating FGFRs in neurons and thatlocalized changes in calcium in growth cones triggerthis response.

UPSTREAM FROM CALCIUM INFLUX

Biochemical and pharmacological studies have pro-vided insights into the nature of the cascade that cou-ples activation of the FGFR to calcium influx intogrowth cones. Figure 2 illustrates a FGFR 3 PLCg 3DAG lipase-dependent signaling cascade that is re-quired for the neurite outgrowth response stimulatedby CAMs and FGF2 (Archer et al., 1999; Lom et al., 1998;Meiri et al., 1998; Ronn et al., 1999, 2000; Walsh andDoherty, 1997). The key enzymatic steps are activationof PLCg to generate DAG and the conversion of DAG toarachidonic acid by DAG lipase. DAG lipase activity isalso essential for both the L1 and FGF2 stimulation ofcalcium influx into growth cones (Archer et al., 1999).This step is pivotal in the FGFR pathway as arachidonicacid can fully mimic the FGF response in a calcium-dependent manner in primary neurons (Williams et al.,1994c). Interestingly, arachidonic acid is also a set-pointmessenger. Increasing arachidonic acid levels towardthe set-point stimulates axonal growth, whereas in-creasing it above the set-point inhibits axonal growthand this allows the FGFR to stimulate or inhibit axonalgrowth depending on the nature of the substrate thatneurons are growing on (Williams et al., 1995b). Ingeneral, L- and/or N-type calcium channel antagonistscan inhibit calcium influx and neurite outgrowth re-sponses stimulated by CAMs and FGF (Archer et al.,1999). However, when neurons are grown over highlevels of substrate bound L1-Fc the neurite outgrowthresponse can be inhibited by antibodies to the FGFR,the DAG lipase inhibitor, an agent that buffers intracel-lular calcium and a Ca21/calmodulin kinase inhibitor,but is not inhibited by a combination of N- and L-typecalcium channel antagonists (Williams et al., 1995b).

his suggests that under some circumstances arachi-onic acid can stimulate calcium entry through addi-

ional pathways. Interestingly, in some circumstancesurified N-cadherin can also trigger calcium influx intorowth cones through other channels (Bixby et al.,994). It will be of interest to determine if PLCg, DAG

lipase and arachidonic acid play a role in coupling any
of the other extracellular cues to calcium influx intogrowth cones, particularly in light of the observation
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that inhibition of DAG lipase can perturb axonal path-finding in the developing retina (Brittis et al., 1996) andubstantially inhibit axonal growth in the optic tractLom et al., 1998). In this context, it is noteworthy thatctivation of PLCg is essential for triggering a growth

cone turning response in an NGF gradient, a responsethat also involves calcium influx into growth cones(Ming et al., 1999).

DOWNSTREAM FROM CALCIUM INFLUX

Localized changes in calcium concentration ingrowth cones can be viewed as a pivotal step in thepathway that controls axonal growth and guidancestimulated by many agents including neurotransmitters(Goldberg, 1998; Mattson, 1988; Mattson and Kater,1987; Zheng et al., 1994), electrical fields (McCaig, 1989),nerve activity (Fields et al., 1990), CAMs (Archer et al.,1999; Doherty et al., 1991; Williams et al., 1992), laminintransients (Bixby et al., 1994; Kuhn et al., 1998) netrinsHong et al., 2000; Ming et al., 1997), and FGF (Archer etl., 1999; Williams et al., 1994b). It seems likely thatalcium will couple many of these stimuli to commonffectors that will serve to decode the calcium signal. Its well established that the ultimate target of calcium ishe actin microfilaments that are enriched in the P-omain of the growth cone (e.g., see Lankford andetourneau, 1989; Ohbayashi et al., 1998; Welnhofer etl., 1999; reviewed in Gordon-Weeks, 2000), the path-ays linking changes in calcium to changes in the dy-

amics of actin polymerization are less clear.In the context of the CAM/FGFR cascade illustrated

n Fig. 2, activation of the cascade stimulates phosphor-lation of GAP43 in isolated growth cones and GAP43unction is required for the neurite outgrowth responseMeiri et al., 1998). Importantly, GAP43 is highly con-entrated in the cortical cytoskeleton, and can be mem-rane tethered consequential to dynamic palmitoyl-tion at the N-terminus (reviewed in Gordon-Weeks,000). This makes GAP43 an ideal candidate for re-ponding to localized changes in calcium. GAP43 haseen shown to be important for some aspects of axonalrowth and guidance in vivo (Sretavan and Kruger,998; Strittmatter et al., 1995). However, it should beoted that at least two other molecules that are ex-ressed in neurons can substitute functionally forAP43, and these are the MARKS protein and CAP23

Frey et al., 2000; Laux et al., 2000). Furthermore, calciumnflux into growth cones through L-type calcium chan-

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nels has been shown to result in the phosphorylation ofboth GAP43 and the MARKS protein (Obayashi et al.,1998). Thus, all three members of this “functional” fam-ily are likely to be key effectors for decoding calciumsignals in growth cones. Moreover, GAP43, CAP23 andMARKS have the potential to directly modulate growthcone motility as all three can directly modulate actindynamics (Frey et al., 2000; He et al., 1997; Laux et al.,000). In addition, phosphorylation of this class of mol-cule by PKC results in the release of calmodulin (re-iewed in Skene, 1990) (see Fig. 2). A combination ofalcium influx and free calmodulin should serve toctivate Ca21/calmodulin kinase in the growth cone,

and this is of interest as Ca21/calmodulin kinase inhib-itors inhibit the neurite outgrowth response stimulatedby CAMs and FGF at a step downstream from calciuminflux (Lom et al., 1998; Williams et al., 1995a), as well asneurite outgrowth stimulated by depolarization (Solemet al., 1995) and laminin transients (Kuhn et al., 1998).Thus, the GAP43/MARKS/CAP23 class of moleculemost likely work “hand-in hand” with Ca21/calmodu-in kinase to regulate axonal growth and guidance. LikeAP43 (see above) the Ca21/calmodulin kinase(s) haveeen shown to be important for axonal growth anduidance in vivo (Brittis et al., 1996; Lom et al., 1998;anBerkum and Goodman, 1995)

A SPECIFIC ROLE MAPK IN THE CAMPATHWAY?

The role of the MAPK cascade in neurite outgrowthhas been extensively studied in the PC12 neuronal cellline, and to a lesser extent in primary neurons. Giventhe fact that these models are quite different, we willconsider the data separately.

The MAPK cascade has long been recognized as be-ing required for growth factor induced differentiationof naive PC12 cells (e.g., Pang et al., 1995; Qui andGreen, 1992). More recently, inhibition of the MAPKcascade has been shown to inhibit NCAM stimulatedneurite outgrowth from PC12 cells cultured on NCAMexpressing fibroblasts (Kolkova et al., 2000). Further-more, activation of this cascade by over expression ofactive components of the MAPK cascade can triggerneurite outgrowth from naive PC12 cells (Fukuda et al.,1995; Kolkova et al., 2000). Given that growth factorsand CAMs can activate the MAPK cascade in PC12 cells
(e.g., see Kolkova et al., 2000; Qui and Green, 1992) itppears to be reasonable to conclude that this pathway
is both necessary and sufficient to account for thegrowth factor and CAM responses. However, expres-sion of constitutively active components of the MAPKcascade equates with an oncogenic rather than physio-logical stimulus, and there is no evidence that activationof this cascade by a CAM or growth factor is equivalentto oncogenic signaling. In fact, there is ample evidencethat growth factor activation of the MAPK cascade isnot sufficient for a neurite outgrowth response (Inagakiet al., 1995; Peng et al., 1995; Stephens et al., 1994; Vail-lancourt et al., 1995). Furthermore, following differenti-ation of naive PC12 cells to a sympathetic neuron likecell, neurite outgrowth stimulated by NGF is not de-pendent on the MAPK cascade (Sano and Kitajima,1998). Thus, activation of the MAPK cascade appears tobe a permissive signal involved in making naive PC12cells competent to extend neurites in response to agrowth factor stimulus. It should be noted that naivePC12 cells are also not competent to mount a neuriteoutgrowth response to calcium influx (Doherty et al.,1993; Solem et al., 1995). However, provided the cellsare cultured on a 3T3 cell substrate (Doherty et al., 1993)or there is a subthreshold activation of the NGF recep-tor (Solem et al., 1995), calcium influx can trigger asubstantial neurite outgrowth response. Likewise, sub-threshold signals from 3T3 cells are also required forCAM stimulated neurite outgrowth from naive PC12cells, as naive PC12 cells do not extend neurites whenplated on purified NCAM or L1 (Doherty lab, unpub-lished observation). We would suggest that these sub-threshold signals might work via the MAPK cascade,and that the activation of this cascade is required tomake naive PC12 cells competent to respond to CAMsand calcium.

Inhibition of the MAPK cascade inhibits neurite out-growth by 50–60% when primary neurons are culturedover substrates coated with purified N-cadherin, lami-nin (Perron and Bixby, 1999) or L1 (Schmid et al., 2000).Furthermore, the Bixby lab have shown that ERK2 istransiently activated following plating of chick retinalneurons on N-cadherin and laminin substrates (Perronand Bixby, 1999) and the Maness lab have providedevidence for the transient activation of ERK2 in primarycerebellar granule cells following clustering of L1 orNCAM (Schmid et al., 1999, 2000). Using a neuronal cellline the Maness lab have provided evidence that L1response is mediated by a src 3 PI 3-kinase 3 rac 3MEK1 pathway, whereas the NCAM response dependson a fyn 3 FAK 3 ras 3 rho 3 MEK pathway.

The recent results from the Lemmon and Maness labshave led to new insights on where L1 actually activates

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the MAPK cascade in cells (Schaefer et al., 1999; Schmidt al., 1999). Remarkably, both studies have demon-trated that L1 endocytosis is required for the response,nd it has also been shown by the Lemmon lab that thectivated ERK localizes with a subpool of L1 positivendocytosed vesicles. This result is particularly chal-enging in view of the fact that the Lemmon lab havehown that endocytosis of L1 takes place exclusivelyithin the microtubule rich C-domain of the growth

one rather than the leading edge (Kamiguchi and Lem-

FIG. 3. The FGFR2:FGF1 binding interface from the 1DJS crystalmolecules, which have been separated and rotated through 90 relativthat make close contacts are highlighted in the two crystals, with thillustrate the main interactions between these sequences that underpon the nature of the interaction between the HAV region of the FGFRKyte–Doolittle hydrophobic profile of the HAV region (MEKRLHADoolittle, 1982) and this is shown as the red line in (c). We used the pfor a hydrophobic complement of this sequence within FGF1. The mmotif, and this profile is shown in blue in (c).

on, 2000; Kamiguchi et al., 1998). One consequence of1 endocytosis in growth cones is the cycling of L1 from I

the rear of the growth cone back to the leading edgeand, as suggested by the authors, ERK activity might berequired for the trafficking of L1 in the growth cone. Inthis context, CAMs at the rear of the growth cone mayplay a more “adhesive” role and these CAMs mightneed to be internalized in a MAPK-dependent mannerin order for the growth cone to advance. In support, aMAPK-dependent internalization of the transmem-brane isoform of apCAM (the aplysia homologue ofNCAM) appears to be an early obligatory step in the

wn in (a). The figure shows the solvent accessible surfaces of theach other to expose the binding sites. A number of linear sequences

ual sequences given (in the same color code) in (b). The lines in (b)binding of FGF to the FGFR. In (c) we have focussed our attention

the YCSNGGHF site on FGF1 (both shown in red). We calculated theANTVK) using a linear averaging over 7 amino acids (Kyte and

ples of the molecular recognition theory (Blalock, 1995) and searchedmplementary sequence was the inverse of the LYCSNGGHFLRILPD

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growth of new synaptic connections (Bailey et al., 1997).n this scheme MAPK function is related to its ability to

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phosphorylate apCAM. Interestingly, L1 is also a sub-strate for a number of enzymes associated with theMAPK cascade, including ERK2, and phosphorylationof L1 is associated with L1 internalization (see Schaeferet al., 1999). It should be noted that it has not as yet beendetermined whether inhibition of the MAPK kinasecascade inhibits neurite outgrowth by preventing L1cycling in growth cones, or whether the MAPK cascadeis required for a more general function. In this context,results from the Maness lab suggest that MAPK activityis not required for L1 endocytosis (Schmid et al., 1999).

The alternative view is that CAM activation of theMAPK cascade is an instructive signal that can directlydrive an axonal growth response (Kolkova et al., 2000;Schmid et al., 1999, 2000). In this context, expression ofactive MEK can stimulate neurite outgrowth in primaryneurons (Dimitropoulou and Bixby, 2000). However,the evidence on localization of activated ERK with L1positive vesicles would suggest that activation of ERK2through an L1-initiated mechanism is unlikely to beequivalent to oncogenic activation (see above). In thiscontext, there is also ample evidence that growth factorinduced activation of the MAPK cascade is not suffi-cient for a neurite outgrowth response (see above).Thus, there is no direct evidence in support of the viewthat CAM activation of the MAPK cascade is sufficientto drive an axonal growth response. Interestingly, acti-vation of the Shc-MAPK pathway is also not requiredfor a growth cone turning response in an NGF gradient(Ming et al., 1999).

If an L13 src 3 endocytosis 3 PI 3-kinase 3 rac 3EK1 pathway can directly stimulate a neurite out-

rowth response, then activation of the endogenousool of PI 3-kinase would be expected to mimic theeurite outgrowth response stimulated by L1. We haveecently tested this by treating cerebellar neurons withpeptide that can activate the endogenous pool of PI

-kinase in cells and by doing so fully mimic growthactor stimulated mitogenesis and neuronal survivalDerossi et al., 1998; Williams and Doherty, 1999). Thiseptide did not promote neurite outgrowth from cere-ellar granule cells (E.W., unpublished observation). Inddition, selective activation of the endogenous pool ofI 3-kinase in PC12 cells can fully mimic an NGF sur-ival response, but does not drive an appropriate neu-ite outgrowth response (Ashcroft et al., 1999). Further-

more, a mutated NGF receptor that retains its ability toactivate the PI 3-Kinase cascade, loses its ability tostimulate neurite outgrowth (Peng et al., 1995). Finally,
ecent pharmacological studies have shown that al-hough PI 3-kinase activity is required for growth cone
uidance, it is not required for neurite extension (Mingt al., 1999). Thus there is no evidence to support theiew that activation of the endogenous pool of PI 3-ki-ase is sufficient to drive a neurite outgrowth response.

IS PKC AN EARLY SIGNALINGMOLECULE IN THE NCAM PATHWAY?

Despite the inherent difficulties in translating resultsform naive PC12 cells to primary neurons (see above),recent studies have tried to gain additional insights intothe CAM pathway by culturing these cells on controland NCAM expressing fibroblasts. In addition to con-firming that the NCAM response is absolutely depen-dent on activation of the FGFR (Kolkova et al., 2000), thegroup demonstrated that the protein kinase C specific(sic) inhibitor calphostin C can inhibit the NCAM re-sponse. On the basis of this result, they suggest thatNCAM can promote neurite outgrowth via a novelNCAM-FGFR3 PLCg3 DAG3 PKC3MAPK path-way. In interpreting their data and challenging thescheme outlined in Fig. 2, the authors fail to take intoaccount that in addition to inhibiting PKC, calphostin Cpotently blocks L-type calcium channels (Hartzell andRinderknecht, 1996) and a number of other enzymes incells (e.g., see Lee and Yang, 1996; Redman et al., 1995).Furthermore, if the NCAM-FGFR 3 PLCg 3 DAG 3PKC scheme was correct, inhibition of DAG lipasewould be expected to potentiate CAM responses asinhibition of this enzyme leads to DAG accumulation incells (Kaneki et al., 1998; Freeman, 2000). This is clearlynot the case (Lom et al., 1998; Williams et al., 1994c). Weconclude that there is as yet no substantive evidence forPKC functioning upstream from calcium influx in theCAM/FGFR signaling pathway (although PKCbII islikely to function downstream, see Fig. 2).

N-CADHERIN BINDS TO THEFGFR—INSIGHTS INTO THE BINDINGMECHANISM FROM FGFR CRYSTALS

The results described above clearly show thatNCAM, N-cadherin, and L1 can activate an FGFR-de-pendent signaling cascade in neuronal growth cones.Interactions between N-cadherin and the FGFR havealso been shown to mediate the cell-contact-dependent
survival of ovarian cells (Peluso, 1997), and been impli-cated in the migration of human breast cell cancer lines

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(Hazan et al., 2000; Nieman et al., 1999). Very recently,-cadherin has been shown to co-immunoprecipitateith the FGFR from lysates of ovarian cells (Peluso,

000), and we have confirmed this interaction in a num-er of other cell types (Fiona Howell, Elena Sanchez-eras and PD, unpublished observation). A number of

ecent crystal structures of the FGFR have allowed for ae-evaluation of a theoretical model that first predicteddirect interaction between N-cadherin and the FGFR.The FGFR contains a ;20-amino-acid sequence that

hares homology with sequences found in NCAM, N-adherin and L1 (Williams et al., 1994a). Two experi-ental observations suggest that this region holds the

ey to understanding how CAMs activate the FGFR.irstly, antibodies directed against this region inhibiteurite outgrowth responses stimulated by NCAM, N-adherin and L1 (Williams et al., 1994a). Secondly, pep-ide mimetics of this region from the FGFR (e.g.,MEKKLHAVPAAK) also inhibit the CAM responses

Doherty et al., 1995a; Ronn et al., 1999; Williams et al.,994a). An unexpected set of results in our early studyas the observation that peptides from this region of

he FGFR were shown to be effective inhibitors of FGF-timulated neurite outgrowth. However, there are nownumber of published crystals of FGFR:FGF complexes

hat suggest an explanation for this result (Plotnikov etl., 1999, 2000). Figure 3a shows the structure of theGFR2:FGF1 complex, with the FGF peeled away inrder to highlight a number of linear contact sites (pro-ein accession code 1DJS). What can be seen is that fourinear motifs in FGF1 interact with five linear motifs inhe FGFR2 (Fig. 3b). An essentially identical schemeccounts for the interaction between FGF2 and theGFR1 (protein accession code 1CVS). What is intrigu-

ng is that the HAV region of FGFR (EKRLHAVPAANn the FGFR2) makes extensive contacts with a smallinear motif in the FGFs (the YCSNGGHF motif inGF1). It now seems likely the peptide mimetics of thisegion inhibit FGF function by binding to FGF andirectly modulating the ligand receptor interaction.urther analysis shows that the binding of FGF to theAV region of the FGFR can be explained by comple-entary hydrophobic interactions (Fig. 3c). In fact, if

he hydrophobic profile of the MEKRLHAVPAANTVKequence of FGR2 is used as an in silico probe, it cor-ectly predicts the LYCSNGGHFLRILPD motif in FGF1s an interacting site based on the fact that this is theest hydrophobic complement.Interestingly, recent peptide competition studies sug-

est that homophilic binding between N-cadherin mol-cules depends upon reciprocal interactions between an

AV containing region on one monomer (Williams etl., 2000a) with an INPISGQ motif in the second mono-er (Williams et al., 2000b). It is important to note that

he HAV motif itself plays a minor role in the interac-ion, with most of the binding energy being contributedy the amino acids that immediately flank this motif.urthermore, as with the FGF:FGFR interaction, com-lementary hydrophobicity plays a role in the binding.ased on sequence homology to the N-cadherin motif

n ECD1 that interacts with the N-cadherin HAV re-ion, we have previously suggested that the IDPVNGQotif in ECD4 of N-cadherin might directly interactith the HAV region of the FGFR (Doherty and Walsh,

996). To test the feasibility of this postulate in the lightf the new crystal structures, we have performed aecond in silico experiment. When the hydrophobic pro-le of the FGFR HAV region (MEKRLHAVPAANTVK)

s probed against the complete extracellular region of-cadherin, it selects the LKIDPVNGQITTIAV motif in

ECD4 as the best hydrophobic complement (with theunderlined sequence being the one that we previouslysuggested might serve as an FGFR binding site). It willbe of interest to determine if the recently reported bind-ing interaction between N-cadherin and the FGFR (Pe-luso, 2000) is in fact mediated by the N-cadherin ECD4motif interacting with the HAV region of the FGFR.

CONCLUSION

Recent studies have established calcium as a pivotalsecond messenger that can control axonal guidance inan instructive manner by acting in localized regions ofthe growth cone. Studies on a number of CAMs haveled to the elucidation of a signaling cascade that cancouple an extracellular recognition event to a localizedchange in growth cone calcium levels. Localized cal-cium influx has the potential to phosphorylate GAP43,CAP23, and MARKS via a PKC-dependent mechanismand to activate the Ca21/calmodulin kinase(s). Theseeffectors are likely to work “hand-in-hand” to controlgrowth cone motility by directly modulating the kinet-ics of actin polymerization. An emerging role for theMAPK cascade in the control of axonal growth andguidance must be viewed with more caution. Atpresent, the evidence points to a permissive rather thanan instructive role, and it is clear that CAMs are still ableto promote substantial neurite outgrowth when thispathway is inhibited. Finally, a direct CAM/FGFR in-
teraction has been proposed as the most likely mecha-nism to account for the clear interplay between CAMs

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and the FGFR in the control of axonal growth. Recentstudies show that N-cadherin will coprecipitate withthe FGFR, and structural studies on the FGFR supportthe hypothesis of a direct N-cadherin–FGFR interaction.

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Received August 17, 2000Accepted August 21, 2000

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CAMs and Axonal Growth: A Critical Evaluation of the Role of Calcium and the MAPK Cascade