Activation of EGF receptor kinase by L1-mediated homophilic cell interactions

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.     

Phosphorylation of L1-type cell-adhesion molecules--ankyrins away!

Neural cell-adhesion molecules (CAMs) are powerful initiators of neurite outgrowth during neural differentiation. In addition, their interactions with cytoskeletal components are often conserved and highly regulated. How these two aspects of neural CAM function relate to each other is not well understood. However, a recent publication from the Felsenfeld laboratory ( http://www.mssm.edu/labs/felsenfeld) fills a gap in our knowledge of how the interaction of L1-type CAMs with the membrane skeleton adaptor protein ankyrin is severed by phosphorylation and suggests a feedback mechanism whereby the neurite-stimulating activity of L1-CAM is inversely connected to its cytoskeleton binding.     

The Production of Arachidonic Acid Can Account for Calcium Channel Activation in the Second Messenger Pathway Underlying Neurite Outgrowth Stimulated by NCAM, N-Cadherin, and L1

Abstract: We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)—NCAM, N-cadherin, and L1—as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N-and l -type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 μ M ) and that this response can be inhibited by N-and l -type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A₂ or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage-dependent calcium currents in cardiac myocytes.     

Localized role of CRMP1 and CRMP2 in neurite outgrowth and growth cone steering

Collapsin response mediator protein 1 (CRMP1) and CRMP2 have been known as mediators of extracellular guidance cues such as semaphorin 3A and contribute to cytoskeletal reorganization in the axonal pathfinding process. To date, how CRMP1 and CRMP2 focally regulate axonal pathfinding in the growth cone has not been elucidated. To delineate the local functions of these CRMPs, we carried out microscale‐chromophore‐assisted light inactivation (micro‐CALI), which enables investigation of localized molecular functions with highly spatial and temporal resolutions. Inactivation of either CRMP1 or CRMP2 in the neurite shaft led to arrested neurite outgrowth. Micro‐CALI of CRMP2 in the central domain of the growth cones consistently arrested neurite outgrowth, whereas micro‐CALI of CRMP1 in the same region caused significant lamellipodial retraction, followed by retardation of neurite outgrowth. Focal inactivation of CRMP1 in its half region of the growth cone resulted in the growth cone turning away from the irradiated site. Conversely, focal inactivation of CRMP2 resulted in the growth cone turning toward the irradiated site. These findings suggest different functions for CRMP1 and CRMP2 in growth cone behavior and neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

unc-44 Ankyrin and stn-2 gamma-syntrophin regulate sax-7 L1CAM function in maintaining neuronal positioning in Caenorhabditis elegans

The L1 family of single-pass transmembrane cell adhesion molecules (L1CAMs) is conserved from Caenorhabditis elegans and Drosophila to vertebrates and is required for axon guidance, neurite outgrowth, and maintenance of neuronal positions. The extracellular region of L1CAMs mediates cell adhesion via interactions with diverse cell-surface and extracellular matrix proteins. In contrast, less is known regarding the function of the intracellular domains in the L1CAM cytoplasmic tail. Previously, we identified a role of the C. elegans L1CAM homolog, SAX-7, in maintaining neuronal and axonal positioning. Here, we demonstrate that this function is dependent on three conserved motifs that reside in the SAX-7 cytoplasmic tail: (1) the FERM-binding motif, (2) the ankyrin-binding domain, and (3) the PDZ-binding motif. Furthermore, we provide molecular and genetic evidence that UNC-44 ankyrin and STN-2 γ-syntrophin bind SAX-7 via the respective ankyrin-binding and PDZ-binding motifs to regulate SAX-7 function in maintaining neuronal positioning.     

Shootin1 interacts with actin retrograde flow and L1-CAM to promote axon outgrowth

Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow-CAM linkage model ("clutch" model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.     

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng‐CAM

Mammalian L1 and avian Ng‐CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng‐CAM. Our results indicate that Ig domains 1–4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng‐CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1–4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1–6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 287–302, 2000     

Critical and optimal Ig domains for promotion of neurite outgrowth by L1/Ng-CAM

Mammalian L1 and avian Ng-CAM are homologous neural cell adhesion molecules (CAMs) that promote neurite outgrowth and cell adhesion in most neurons. Previous attempts to map these activities to discrete regions in the CAMs have suggested the involvement of a variety of different domains. However, these studies mainly used bacterially expressed proteins that were much less active on a molar basis than the native molecules. To define regions that are critical for maximal neurite outgrowth, we constructed and tested a panel of eukaryotically expressed proteins containing various extracellular segments of human L1 (hL1) or Ng-CAM. Our results indicate that Ig domains 1-4 of hL1 are critical for homophilic binding and neurite outgrowth; however this segment is less potent than the entire extracellular region. Optimal neurite outgrowth activity was seen with proteins containing all six Ig domains of hL1 or Ng-CAM. The adhesive properties of hL1 fragments correlated tightly with their neurite outgrowth activities, suggesting that these two processes are closely linked. These results suggest that Ig domains 1-4 form a structural cassette responsible for hL1 homophilic binding, while Ig domains 1-6 represent a functional region for optimal promotion of neurite outgrowth in vitro and possibly in vivo.     

L1 and NCAM adhesion molecules as signaling coreceptors in neuronal migration and process outgrowth

Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily engage in multiple neuronal interactions that influence cell migration, axonal and dendritic projection, and synaptic targeting. Their downstream signal transduction events specify whether a cell moves or projects axons and dendrites to targets in the brain. Many of the diverse functions of CAMs are brought about through homophilic and heterophilic interactions with other cell surface receptors. An emerging concept is that CAMs act as coreceptors to assist in intracellular signal transduction, and to provide cytoskeletal linkage necessary for cell and growth cone motility. Here we will focus on new discoveries that have revealed novel coreceptor functions for the best-understood CAMs--L1, CHL1, and NCAM--important for neuronal migration and axon guidance. We will also discuss how dysregulation of CAMs may also bear on neuropsychiatric disease and cancer.     

Activity-dependent regulation of N-cadherin in DRG neurons: Differential regulation of N-cadherin,NCAM, and L1 by distinct patterns of action potentials

Cell adhesion molecule (CAM) expression is highly regulated during nervous system development to control cell migration, neurite outgrowth, fasciculation, and synaptogenesis. Using electrical stimulation of mouse dorsal root ganglion (DRG) neurons in cell culture, this work shows that N-cadherin expression is regulated by neuronal firing, and that expression of different CAMs is regulated by distinct patterns of neural impulses. N-cadherin was down-regulated by 0.1 or 1 Hz stimulation, but NCAM mRNA and protein levels were not altered by stimulation. L1 was down-regulated by 0.1 Hz stimulation, but not by 0.3 Hz, 1 Hz, or pulsed stimulation. N-cadherin expression was lowered with faster kinetics than L1 (1 vs. 5 days), and L1 mRNA returned to higher levels after terminating the stimulus. The RSLE splice variant of L1 was not regulated by action potential stimulation, and activity-dependent influences on L1 expression were blocked by target-derived influences. The results are consistent with changes in firing pattern accompanying DRG development and suggest that functional activity can influence distinct developmental processes by regulating the relative abundance of different CAMs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 735–748, 1997

1 This is a US Government work and, as such, is in the public domain in the United States of America.

     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2 mediated axon guidance

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilin1, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1CAMs.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

The RNA Binding Protein Igf2bp1 Is Required for Zebrafish RGC Axon Outgrowth In Vivo

Attractive growth cone turning requires Igf2bp1-dependent local translation of β-actin mRNA in response to external cues in vitro. While in vivo studies have shown that Igf2bp1 is required for cell migration and axon terminal branching, a requirement for Igf2bp1 function during axon outgrowth has not been demonstrated. Using a timelapse assay in the zebrafish retinotectal system, we demonstrate that the β-actin 3’UTR is sufficient to target local translation of the photoconvertible fluorescent protein Kaede in growth cones of pathfinding retinal ganglion cells (RGCs) in vivo. Igf2bp1 knockdown reduced RGC axonal outgrowth and tectal coverage and retinal cell survival. RGC-specific expression of a phosphomimetic Igf2bp1 reduced the density of axonal projections in the optic tract while sparing RGCs, demonstrating for the first time that Igf2bp1 is required during axon outgrowth in vivo. Therefore, regulation of local translation mediated by Igf2bp proteins may be required at all stages of axon development.     

Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4)

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.     

A Phylogenetic Analysis of the L1 Family of Neural Cell Adhesion Molecules

L1-type genes form one of several distinct gene families that encode adhesive proteins, which are predominantly expressed in developing and mature metazoan nervous systems. These proteins have a multitude of different important cellular functions in neuronal and glial cells. L1-type gene products are transmembrane proteins with a characteristic extracellular domain structure consisting of six immunoglobulin and three to five fibronectin type III protein folds. As reported here, L1-type proteins can be identified in most metazoan phyla with the notable exception of Porifera (sponges). This puts the origin of L1-type genes at a point in time when primitive cellular neural networks emerged, approximately 1,200 to 1,500 million years ago. Subsequently, several independent gene duplication events generated multiple paralogous L1-type genes in some phyla, allowing for a considerable diversification of L1 structures and the emergence of new functional features and molecular interactions. One such evolutionary newer feature is the appearance of RGD integrin-binding motifs in some vertebrate L1 family members.     

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. In this study, we describe the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. We uncover a novel role for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1.     

Cis and trans interactions of L1 with neuropilin-1 control axonal responses to semaphorin 3A

Mutations in the L1 gene induce a spectrum of human neurological disorders due to abnormal development of several brain structures and fiber tracts. Among its binding partners, L1 immunoglobulin superfamily adhesion molecule (Ig CAM) associates with neuropilin-1 (NP-1) to form a semaphorin3A (Sema3A) receptor and soluble L1 converts Sema3A-induced axonal repulsion into attraction. Using L1 constructs containing missense pathological mutations, we show here that this reversion is initiated by a specific trans binding of L1 to NP-1, but not to L1 or other Ig CAMs, and leads to activation of the NO/cGMP pathway. We identified the L1-NP-1-binding site in a restricted sequence of L1 Ig domain 1, as a peptide derived from this region could reverse Sema3A repulsive effects. A pathological L1 missense mutation located in this sequence specifically disrupts both L1-NP-1 complex formation and Sema3A reversion, suggesting that the cross-talk between L1 and Sema3A might participate in human brain development.     

Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms

It has recently become clear that both extracellular matrix (ECM) glycoproteins and various cell adhesion molecules (CAMs) can promote neurite outgrowth from primary neurons, though little is known of the intracellular mechanisms through which these signals are transduced. We have previously obtained evidence that protein kinase C function is an important part of the neuronal response to laminin (Bixby, J.L. 1989. Neuron. 3:287-297). Because such CAMs as L1 (Lagenauer, C., and V. Lemmon. 1987. Proc. Natl. Acad. Sci. USA. 84:7753-7757) and N-cadherin (Bixby, J.L. and R. Zhang. 1990. J. Cell Biol. 110:1253-1260) can be purified and used as substrates to promote neurite growth, we have now tested whether the response to CAMs is similarly dependent on protein kinase C. We find that inhibition of protein kinase C inhibits growth on fibronectin or collagen as well as on laminin. In contrast, C kinase inhibition actually potentiates the initial growth response to L1 or N- cadherin. The later "phase" of outgrowth on both of these CAMs is inhibited, however. Additionally, phorbol esters, which have no effect on neurite growth when optimal laminin concentrations are used, potentiate growth even on optimal concentrations of L1 or N-cadherin. The results indicate that different intracellular mechanisms operate during initial process outgrowth on ECM substrates as compared to CAM substrates, and suggest that protein kinase C function is required for continued neurite growth on each of these glycoproteins.