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.     

Neural integrity is maintained by dystrophin in C. elegans

The dystrophin protein complex (DPC), composed of dystrophin and associated proteins, is essential for maintaining muscle membrane integrity. The link between mutations in dystrophin and the devastating muscle failure of Duchenne's muscular dystrophy (DMD) has been well established. Less well appreciated are the accompanying cognitive impairment and neuropsychiatric disorders also presented in many DMD patients, which suggest a wider role for dystrophin in membrane-cytoskeleton function. This study provides genetic evidence of a novel role for DYS-1/dystrophin in maintaining neural organization in Caenorhabditis elegans. This neuronal function is distinct from the established role of DYS-1/dystrophin in maintaining muscle integrity and regulating locomotion. SAX-7, an L1 cell adhesion molecule (CAM) homologue, and STN-2/γ-syntrophin also function to maintain neural integrity in C. elegans. This study provides biochemical data that show that SAX-7 associates with DYS-1 in an STN-2/γ-syntrophin-dependent manner. These results reveal a recruitment of L1CAMs to the DPC to ensure neural integrity is maintained.     

The interaction between L1-type proteins and ankyrins - a master switch for L1-type CAM function

L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that ankyrin-binding to L1-type CAMs provides a master switch. The interaction with ankyrins directs L1-type adhesive proteins into different functional contexts, either ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into ankyrin-dependent functions, such as L1’s role at axon initial segments (AIS), paranodal regions, synapses and in dendrites. The content of this Mini review was first presented in a shortened form at the 12th Mejbaum-Katzenellenbogen Seminar “Membrane Skeleton. Recent Advances and Future Research Directions”, June 15–18, 2008, Zakopane, Poland     

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.     

A role for the C. elegans L1CAM homologue lad-1/sax-7 in maintaining tissue attachment

The L1 family of cell adhesion molecules (L1CAMs) is important for neural development. Mutations in one of the human L1CAM genes, L1, can result in several neurological syndromes, the symptoms of which are variably penetrant. The physiological cause of these symptoms, collectively termed CRASH, is not clear. Caenorhabditis elegans animals genetically null for the L1CAM homologue LAD-1, exhibit variably penetrant pleiotropic phenotypes that are similar to the CRASH symptoms; thus the C. elegans lad-1 mutant provides an excellent model system to study how disruption of L1 leads to these abnormalities. These phenotypes include uncoordinated movements, variable embryonic lethality, and abnormal neuronal distribution and axon trajectories. Our analysis revealed that many of these phenotypes are likely a result of tissue detachment.     

Constitutive internalization of constitutively active agiotensin II AT(1A) receptor mutants is blocked by inverse agonists.

As constitutively active mutants (CAMs) mimic an active conformation, they can be used to characterize the process of G protein-coupled receptor activation. Here, we used CAMs to study the link between activation and internalization of the angiotensin II AT(1A) receptor. The cellular localization of fluorescently tagged N111A, I245T, and L305Q mutants was determined by confocal microscopy. In the absence of ligand, CAMs were mostly located in intracellular vesicles, whereas the wild-type AT(1A) was found at the cell surface. After 2 h incubation with inverse agonist, losartan, CAMs were translocated to the plasma membrane. Similar observations were made in H295, a human adrenocortical cell line which expresses physiologically the AT(1) receptor. This phenomenon, which was not dependent on protein synthesis and the pharmacology and kinetics of which were similar to the recycling of the wild-type receptor, was called "externalization". After externalization and losartan removal, the L305Q CAM underwent rapid ligand-independent endocytosis, with the same kinetics and temperature sensitivity as the angiotensin II-induced internalization of the wild-type AT(1A). Moreover, the addition of a second mutation known to block internalization (Delta 329 truncation) prevented intracellular localization of the CAM. These data show that AT(1A) CAMs are constitutively and permanently internalized and recycled. This mechanism is different from the down-regulation observed for CAMs of other G protein-coupled receptors and thus defines a new paradigm for the cellular regulation of CAMs.     

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments.

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.     

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.

     

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     

The mechanism of axon growth

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities.

Mutations in the gene for neural cell adhesion molecule L1 (L1CAM) result in a debilitating X-linked congenital disorder of brain development. At the neuronal cell surface L1 may interact with a variety of different molecules including itself and two other CAMs of the immunoglobulin superfamily, axonin-1 and F11. However, whether all of these interactions are relevant to normal or abnormal development has not been determined. Over one-third of patient mutations are single amino acid changes distributed across 10 extracellular L1 domains. We have studied the effects of 12 missense mutations on binding to L1, axonin-1 and F11 and shown for the first time that whereas many mutations affect all three interactions, others affect homophilic or heterophilic binding alone. Patient pathology is therefore due to different types of L1 malfunction. The nature and functional consequence of mutation is also reflected in the severity of the resultant phenotype with structural mutations likely to affect more than one binding activity and result in early mortality. Moreover, the data indicate that several extracellular domains of L1 are required for homophilic and heterophilic interactions.     

The mechanism of axon growth: what we have learned from the cell adhesion molecule L1

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

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.     

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.     

Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner

Motility of nerve growth cones (GCs) is regulated by region-specific activities of cell adhesion molecules (CAMs). CAM activities could be modified by their localization to detergent-resistant membranes (DRMs), specialized microdomains enriched in signaling molecules. This paper deals with a question of whether DRMs are involved in GC migration stimulated by three CAMs; L1, N-cadherin (Ncad), and beta1 integrin. We demonstrate that L1 and Ncad are present in DRMs, whereas beta1 integrin is exclusively detected in non-DRMs of neurons and that localization of L1 and Ncad to DRMs is developmentally regulated. GC migration mediated by L1 and Ncad but not by beta1 integrin is inhibited after DRM disruption by micro-scale chromophore-assisted laser inactivation (micro-CALI) of GM1 gangliosides or by pharmacological treatments that deplete cellular cholesterol or sphingolipids, essential components for DRMs. Characteristic morphology of GCs induced by L1 and Ncad is also affected by micro-CALI-mediated DRM disruption. Micro-CALI within the peripheral domain of GCs, or even within smaller areas such as the filopodia and the lamellipodia, is sufficient to impair their migration. However, micro-CALI within the central domain does not affect GC migration. These results demonstrate the region-specific involvement of DRMs in CAM-dependent GC behavior.     

Neural circuit formation in the cerebellum is controlled by cell adhesion molecules of the Contactin family

Cell adhesion molecules of the immunoglobulin superfamily (IgSF CAMs) have been implicated in neural circuit formation in both the peripheral and the central nervous system. Several recent studies highlight a role of the Contactin group of IgSF CAMs in cerebellar development, in particular in the development of granule cells. Granule cells are the most numerous type of neurons in the nervous system and by forming a secondary proliferative zone in the cerebellum they provide an exception to the rule that neuronal precursors proliferate in the ventricular zone. Granule cells express Contactin-2, Contactin-1, and Contactin-6 in a sequential manner. Contactins are required for axon guidance, fasciculation, and synaptogenesis, and thus affect multiple steps in neural circuit formation in the developing cerebellum.     

Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.     

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.