DRG Axon Elongation and Growth Cone Collapse Rate Induced by Sema3A are Differently Dependent on NGF Concentration

Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon’s regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.     

Temporal regulation of neuropilin-1 expression and sensitivity to semaphorin 3A in NGF- and NT3-responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.     

Temporal regulation of neuropilin‐1 expression and sensitivity to semaphorin 3A in NGF‐ and NT3‐responsive chick sensory neurons

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small‐caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large‐muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin‐1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)‐ and NT3 (proprioceptive)‐dependent sensory axons extended from E6‐E10 chick embryos. Growth cones extended from E6 DRGs in NT3‐containing medium expressed neuropilin‐1 and collapsed in response to Sema3A. From E7 until E10 NT3‐responsive growth cones expressed progressively lower levels of neuropilin‐1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF‐containing medium expressed progressively higher levels of neuropilin‐1 and higher levels of collapse response to Sema3A over the period from E6–E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin‐1. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 43–53, 2002     

Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity

The repellent semaphorin 3A (Sema3A) causes growth cone turning or collapse by triggering cytoskeletal rearrangements and detachment of adhesion sites. Growth cone detachment is dependent on eicosanoid activation of protein kinase C epsilon (PKCε), but the characterization of the phospholipase A(2) (PLA(2) ) that releases arachidonic acid (AA) for eicosanoid synthesis has remained elusive. Here, we show, in rat dorsal root ganglion (DRG) neurons, that Sema3A stimulates PLA(2) activity, that Sema3A-induced growth cone turning and collapse are dependent on the release of AA, and that the primary PLA(2) involved is the group IV α isoform (GIVA). Silencing GIVA expression renders growth cones resistant to Sema3A-induced collapse, and GIVA inhibition reverses Sema3A-induced repulsion into attraction. These studies identify a novel, early step in Sema3A-signaling and a PLA(2) necessary for growth cone repulsion and collapse.     

Molecular basis of semaphorin-mediated axon guidance

The semaphorin family of proteins constitute one of the major cues for axonal guidance. The prototypic member of this family is Sema3A, previously designated semD/III or collapsin-1. Sema3A acts as a diffusible, repulsive guidance cue in vivo for the peripheral projections of embryonic dorsal root ganglion neurons. Sema3A binds with high affinity to neuropilin-1 on growth cone filopodial tips. Although neuropilin-1 is required for Sema3A action, it is incapable of transmitting a Sema3A signal to the growth cone interior. Instead, the Sema3A/neuropilin-1 complex interacts with another transmembrane protein, plexin, on the surface of growth cones. Certain semaphorins, other than Sema3A, can bind directly to plexins. The intracellular domain of plexin is responsible for initiating the signal transduction cascade leading to growth cone collapse, axon repulsion, or growth cone turning. This intracellular cascade involves the monomeric G-protein, Rac1, and a family of neuronal proteins, the CRMPs. Rac1 is likely to be involved in semaphorin-induced rearrangements of the actin cytoskeleton, but how plexin controls Rac1 activity is not known. Vertebrate CRMPs are homologous to the Caenorhabditis elegans unc-33 protein, which is required for proper axon morphology in worms. CRMPs are essential for Sema3A-induced, neuropilin-plexin-mediated growth cone collapse, but the molecular interactions of growth cone CRMPs are not well defined. Mechanistic aspects of plexin-based signaling for semaphorin guidance cues may have implications for other axon guidance events and for the basis of growth cone motility.     

Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse

Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.     

R-Type Calcium Channels Are Crucial for Semaphorin 3A–Induced DRG Axon Growth Cone Collapse

Semaphorin 3A (Sema3A) is a secreted protein involved in axon path-finding during nervous system development. Calcium signaling plays an important role during axonal growth in response to different guidance cues; however it remains unclear whether this is also the case for Sema3A. In this study we used intracellular calcium imaging to figure out whether Sema3A-induced growth cone collapse is a Ca²⁺ dependent process. Intracellular Ca²⁺ imaging results using Fura-2 AM showed Ca²⁺ increase in E15 mice dorsal root ganglia neurons upon Sema3A treatment. Consequently we analyzed Sema3A effect on growth cones after blocking or modifying intracellular and extracellular Ca²⁺ channels that are expressed in E15 mouse embryos. Our results demonstrate that Sema3A increased growth cone collapse rate is blocked by the non-selective R- and T- type Ca²⁺ channel blocker NiCl₂ and by the selective R-type Ca²⁺ channel blocker SNX482_. These Ca²⁺ channel blockers consistently decreased the Sema3A-induced intracellular Ca²⁺ concentration elevation. Overall, our results demonstrate that Sema3A-induced growth cone collapses are intimately related with increase in intracellular calcium concentration mediated by R-type calcium channels.     

Fyn and Cdk5 mediate semaphorin-3A signaling,which is involved in regulation of dendrite orientation in cerebral cortex

Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.     

Lipid rafts mediate chemotropic guidance of nerve growth cones

Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.     

Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32.

BACKGROUND: Neurons project their axons along specific pathways in order to establish appropriate connections with their target cells. The rate and direction of axonal growth is determined by interactions between the highly motile growth cone and environmental cues that can act in either an attractive or a repulsive manner. Locomotion is ultimately dependent upon the reorganisation of the actin cytoskeleton and an established role for the Rho family of small GTPases in regulating this process in non-neuronal cells identifies them as candidate signalling molecules in growth cones. An inactive form of Rac1 has recently been shown to inhibit the 'growth-cone collapse' response induced by chick Sema3A, a protein that has recently been established as an important guidance cue. The molecular basis for this inhibition remains unclear. RESULTS: We have made a series of overlapping peptides from the amino-terminal region of Rac1 and rendered them cell permeable by synthesis in tandem with an established internalisation vector. We report here that a peptide encompassing Rac1 amino acids 17-32 binds directly to the established Rac1-interacting molecules PAK, WASP, 3BP-1 and p85beta(P13K), but not to p67(Phox). Furthermore, the peptide can compete with activated Rac1 for target binding, and inhibits Sema3A-induced growth-cone collapse. We also synthesised cell-permeable peptides that correspond to the Cdc42/Rac1-binding (CRIB) motifs present in PAK and N-WASP. Our results show that a CRIB-containing peptide from PAK, but not that from N-WASP, inhibits growth-cone collapse and that the inhibitory activity correlates with binding to Rac1 and not to Cdc42. CONCLUSIONS: Our results suggest that Sema3A-induced growth-cone collapse is mediated by Rac1 amino acids 17-32, and demonstrate the feasibility of designing new cell-permeable inhibitors of small GTPases.     

Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors.

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. We find that the two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.     

Structural bases for CRMP function in plexin-dependent semaphorin3A signaling

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP2 was previously shown to mediate the repulsive effect of Sema3A on axons and to participate in axonal specification. The X-ray crystal structure of murine CRMP1 was determined at 2.1 A resolution and demonstrates that CRMP1 is a bilobed 'lung-shaped' protein forming a tetrameric assembly. Structure-based mutagenesis of surface-exposed residues was employed to map functional domains. As a rapid assay for CRMP, we exploited a reconstituted Sema3A signaling system in COS-7 cells expressing the receptor components Neuropilin1 and PlexinA1 (NP1/PlexA1). In these cells, CRMP and PlexA1 form a physical complex that is reduced in amount by NP1 but enhanced by Sema3A/NP1. Furthermore, CRMP accelerates Sema3A-induced cell contraction. Alanine substitutions in one domain of CRMP1 produce a constitutively active protein that causes Sema3A-independent COS-7 contraction. This mutant CRMP mimics the DRG neurite outgrowth-inhibiting effects of Sema3A and reduces Sema3A-induced axonal repulsion. These data provide a structural view of CRMP function in Plex-dependent Sema3A signaling.     

Dorsal Root Ganglion Neurons React to Semaphorin 3A Application through a Biphasic Response that Requires Multiple Myosin II Isoforms

Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction.     

Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins

The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To overcome the limited number of guidance cues, modulation of axon responsiveness is crucial for specifying accurate trajectories. We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections, among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.     

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.     

Myristoylated, alanine-rich C-kinase substrate phosphorylation regulates growth cone adhesion and pathfinding

Repellents evoke growth cone turning by eliciting asymmetric, localized loss of actin cytoskeleton together with changes in substratum attachment. We have demonstrated that semaphorin-3A (Sema3A)-induced growth cone detachment and collapse require eicosanoid-mediated activation of protein kinase C epsilon (PKC epsilon) and that the major PKC epsilon target is the myristoylated, alanine-rich C-kinase substrate (MARCKS). Here, we show that PKC activation is necessary for growth cone turning and that MARCKS, while at the membrane, colocalizes with alpha3-integrin in a peripheral adhesive zone of the growth cone. Phosphorylation of MARCKS causes its translocation from the membrane to the cytosol. Silencing MARCKS expression dramatically reduces growth cone spread, whereas overexpression of wild-type MARCKS inhibits growth cone collapse triggered by PKC activation. Expression of phosphorylation-deficient, mutant MARCKS greatly expands growth cone adhesion, and this is characterized by extensive colocalization of MARCKS and alpha3-integrin, resistance to eicosanoid-triggered detachment and collapse, and reversal of Sema3A-induced repulsion into attraction. We conclude that MARCKS is involved in regulating growth cone adhesion as follows: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment critical for turning and collapse.     

CHL1 promotes Sema3A-induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane

Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin-dependent neurite outgrowth and semaphorin3A (Sema3A)-mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin-radixin-moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane-proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A-induced growth cone collapse and CHL1-dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A-induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.     

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.     

The activity of the plexin-A1 receptor is regulated by Rac

Plexins constitute a large family of transmembrane proteins that act as receptors for the semaphorin family of ligands. They are best known for their role in growth cone guidance, although they also are widely expressed outside the nervous system. Plexins are thought to control axon guidance by modifying the growth cone cytoskeleton, and Rho GTPases have been strongly implicated in this response. However, the exact contribution of Rho proteins is unclear. Sema3A/Plexin-A1-induced growth cone collapse, for example, requires Rac activity, which is a surprising result given that this GTPase is usually associated with membrane protrusions. We show here that Sema3A-induced collapse of COS-7 cells expressing Plexin-A1 also requires Rac but not Rho activity and that the cytoplasmic tail of Plexin-A1 interacts directly with activated Rac. However, collapse induced by a constitutively activated version of Plexin-A1 does not require Rac. We propose a novel function for Rac, namely that it acts upstream of Plexin-A1 during semaphoring-induced collapse, to regulate the activity of the receptor.