Protein tyrosine phosphatase PTPRT as a regulator of synaptic formation and neuronal development

PTPRT/RPTPρ is the most recently isolated member of the type IIB receptor-type protein tyrosine phosphatase family and its expression is restricted to the nervous system. PTPRT plays a critical role in regulation of synaptic formation and neuronal development. When PTPRT was overexpressed in hippocampal neurons, synaptic formation and dendritic arborization were induced. On the other hand, knockdown of PTPRT decreased neuronal transmission and attenuated neuronal development. PTPRT strengthened neuronal synapses by forming homophilic trans dimers with each other and heterophilic cis complexes with neuronal adhesion molecules. Fyn tyrosine kinase regulated PTPRT activity through phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT. Phosphorylation induced homophilic cis dimerization of PTPRT and resulted in the inhibition of phosphatase activity. BCR-Rac1 GAP and Syntaxin-binding protein were found as new endogenous substrates of PTPRT in rat brain. PTPRT induced polymerization of actin cytoskeleton that determined the morphologies of dendrites and spines by inhibiting BCR-Rac1 GAP activity. Additionally, PTPRT appeared to regulate neurotransmitter release through reinforcement of interactions between Syntaxin-binding protein and Syntaxin, a SNARE protein. In conclusion, PTPRT regulates synaptic function and neuronal development through interactions with neuronal adhesion molecules and the dephosphorylation of synaptic molecules. [BMB Reports 2015; 48(5): 249-255]     

Phosphorylation of Ser 21 in Fyn regulates its kinase activity,focal adhesion targeting,and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010. © 2010 Wiley‐Liss, Inc.     

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.     

EphB receptors interact with NMDA receptors and regulate excitatory synapse formation

EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.     

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.     

Orchestrating the synaptic network by tyrosine phosphorylation signalling

The establishment of a functional brain requires coordinated and stereotyped formation of synapses between neurons. For this, trans-synaptic molecular cues (synaptic organizers) are exchanged between a neuron and its target to organize appropriate synapses. The understanding of signalling mechanisms by which such synaptic organizers lead to synapse formation is just being elucidated. However, recent studies revealed that some of these cues act through receptor protein tyrosine kinases (RPTKs) or phosphatases (RPTPs). Synaptogenic RPTKs and RPTPs pattern synaptic network through affecting local protein-protein binding dynamics, changing the phosphorylation state of signalling cascades, or promoting gene expression. Each RPTK or RPTP has distinct roles in synapse formation, serving at different synapses or showing differential synaptogenic effects. Thus, tyrosine phosphorylation signalling plays critical roles in building the orchestrated synaptic circuitry in the brain.     

Fyn is a downstream target of the pleiotrophin/receptor protein tyrosine phosphatase beta/zeta-signaling pathway: regulation of tyrosine phosphorylation of Fyn by pleiotrophin

Pleiotrophin (PTN the protein, Ptn the gene) signals downstream targets through inactivation of its receptor, the transmembrane receptor protein tyrosine phosphatase (RPTP)beta/zeta, disrupting the balanced activity of RPTPbeta/zeta and the activity of a constitutively active tyrosine kinase. As a consequence of the inactivation of RPTPbeta/zeta, PTN stimulates a sharp increase in the levels of tyrosine phosphorylation of the substrates of RPTPbeta/zeta in PTN-stimulated cells. We now report that the Src family member Fyn interacts with the intracellular domain of RPTPbeta/zeta in a yeast two-hybrid system. We further demonstrate that Fyn is a substrate of RPTPbeta/zeta, and that tyrosine phosphorylation of Fyn is sharply increased in PTN-stimulated cells. In previous studies, we demonstrated that beta-catenin and beta-adducin are targets of the PTN/RPTPbeta/zeta-signaling pathway and defined the mechanisms through which tyrosine phosphorylation of beta-catenin and beta-adducin disrupts cytoskeletal protein complexes. We conclude that Fyn is a downstream target of the PTN/RPTPbeta/zeta-signaling pathway and suggest that PTN coordinately regulates tyrosine phosphorylation of beta-catenin, beta-adducin, and Fyn through the PTN/RPTPbeta/zeta-signaling pathway and that together Fyn, beta-adducin, and beta-catenin may be effectors of the previously described PTN-stimulated disruption of cytoskeletal stability, increased cell plasticity, and loss of cell-cell adhesion that are characteristic of PTN-stimulated cells and a feature of many human malignant cells in which mutations have established constitutive expression of the Ptn gene.     

Dissection of synapse induction by neuroligins: effect of a neuroligin mutation associated with autism

To study synapse formation by neuroligins, we co-cultured hippocampal neurons with COS cells expressing wild type and mutant neuroligins. The large size of COS cells makes it possible to test the effect of neuroligins presented over an extended surface area. We found that a uniform lawn of wild type neuroligins displayed on the cell surface triggers the formation of hundreds of uniformly sized, individual synaptic contacts that are labeled with neurexin antibodies. Electron microscopy revealed that these artificial synapses contain a presynaptic active zone with docked vesicles and often feature a postsynaptic density. Neuroligins 1, 2, and 3 were active in this assay. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta, a presumptive presynaptic binding partner for postsynaptic neuroligins, and blocked synapse formation. An analysis of mutant neuroligins with an amino acid substitution that corresponds to a mutation described in patients with an autistic syndrome confirmed previous reports that these mutant neuroligins have a compromised capacity to be transported to the cell surface. Nevertheless, the small percentage of mutant neuroligins that reached the cell surface still induced synapse formation. Viewed together, our data suggest that neuroligins generally promote artificial synapse formation in a manner that is associated with beta-neurexin binding and results in morphologically well differentiated synapses and that a neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synapse formation activity.     

Protein tyrosine phosphatase alpha regulates Fyn activity and Cbp/PAG phosphorylation in thymocyte lipid rafts

A role for the receptor protein tyrosine phosphatase alpha (PTPalpha) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPalpha-deficient mice. PTPalpha-null thymocytes develop normally, but unstimulated PTPalpha-/- cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPalpha is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPalpha is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPalpha-/- cells. PTPalpha is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPalpha, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPalpha this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPalpha, the long-term proliferative response of PTPalpha-/- thymocytes was reduced. These findings indicate that PTPalpha is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.     

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.     

Fibronectin rigidity response through Fyn and p130Cas recruitment to the leading edge

Cell motility on extracellular matrices critically depends on matrix rigidity, which affects cell adhesion and formation of focal contacts. Receptor-like protein tyrosine phosphatase alpha (RPTPalpha) and the alphavbeta3 integrin form a rigidity-responsive complex at the leading edge. Here we show that the rigidity response through increased spreading and growth correlates with leading edge recruitment of Fyn, but not endogenous c-Src. Recruitment of Fyn requires the palmitoylation site near the N-terminus and addition of that site to c-Src enables it to support a rigidity response. In all cases, the rigidity response correlates with the recruitment of the Src family kinase to early adhesions. The stretch-activated substrate of Fyn and c-Src, p130Cas, is also required for a rigidity response and it is phosphorylated at the leading edge in a Fyn-dependent process. A possible mechanism for the fibronectin rigidity response involves force-dependent Fyn phosphorylation of p130Cas with rigidity-dependent displacement. With the greater displacement of Fyn from p130Cas on softer surfaces, there will be less phosphorylation. These studies emphasize the importance of force and nanometer-level movements in cell growth and function.     

Binding of Fyn to MAP-2c through an SH3 binding domain. Regulation of the interaction by ERK2

Microtubule-associated protein 2 (MAP-2) isoforms are developmentally expressed in the nervous system and contain a number of functional domains. Adjacent to the first repeat of the microtubule-binding domain is an RTPPKSP motif for binding SH3 domains. To identify SH3-containing proteins that interact with MAP-2, transfections, filter overlay assays, glutathione S-transferase (GST)-mediated binding assays, co-immunoprecipitations and enzyme-linked immunosorbent assays were performed. Transfections of MAP-2a, MAP-2b, and MAP-2c constructs into COS7 cells, followed by incubation of the cell lysates with SH3-GST fusion proteins, determined that the strongest interaction was between MAP-2c and the non-receptor tyrosine kinase Fyn; however, MAP-2b and MAP-2c also bound to Grb2. Co-immunoprecipitation of Fyn and MAP-2c from human fetal homogenates confirmed the interaction in vivo. MAP-2 synthetic peptides spanning the RTPPKSP motif bound to Fyn, and the interaction was regulated by phosphorylation. Co-transfections with MAP-2c and the extracellular signal-regulated kinase 2 (ERK2) demonstrated that MAP-2c is threonine/serine-phosphorylated on its RTPPKSP motif and that threonine phosphorylation abolished the MAP-2c/Fyn binding. Kinase assays and co-transfection of MAP-2c and Fyn confirmed that Fyn tyrosine kinase phosphorylates MAP-2c. Thus, the activation of signaling pathways may regulate cytoskeletal dynamics by altering the state of phosphorylation of MAP-2 by both ERK2 and Fyn kinase.     

Neuroligin 2 is exclusively localized to inhibitory synapses

Neuroligins are cell adhesion proteins that are thought to instruct the formation and alignment of synaptic specializations. The three known rodent neuroligin isoforms share homologous extracellular acetylcholinesterase-like domains that bridge the synaptic cleft and bind beta-neurexins. All neuroligins have identical intracellular C-terminal motifs that bind to PDZ domains of various target proteins. Neuroligin 1 is specifically localized to glutamatergic postsynaptic specializations. We show here that neuroligin 2 is exclusively localized to inhibitory synapses in rat brain and dissociated neurons. In immature neurons, neuroligin 2 is found at synapses and also at GABAA receptor aggregates that are not facing presynaptic termini, indicating that postsynaptic mechanisms lead to synaptic recruitment of neuroligin 2. Our findings identify neuroligin 2 as a new cell adhesion protein specific for inhibitory synapses and open new avenues for identifiying the constituents of this unique type of postsynaptic specialization.     

Identification and characterization of a novel Pyk2/related adhesion focal tyrosine kinase-associated protein that inhibits alpha-synuclein phosphorylation

alpha-Synuclein is a presynaptic protein involved in the pathogenesis of several neurodegenerative diseases, such as Parkinson's disease. Pyk2/related adhesion focal tyrosine kinase (RAFTK) tyrosine kinase is an upstream regulator of Src family kinases in the central nervous system that is involved in alpha-synuclein phosphorylation. The present study reports the cloning and characterization of a novel adaptor protein, Pyk2/RAFTK-associated protein (PRAP), that specifically binds to Pyk2/RAFTK and inhibits alpha-synuclein tyrosine phosphorylation. PRAP contains a coiled-coil domain, a pleckstrin homology domain, and a SH3 domain; the SH3 domain binds to the proline-rich domain of Pyk2/RAFTK. PRAP was observed to be present throughout the brain, including substantia nigra dopaminergic neurons, in which it localized to the cytoplasm. PRAP was found to function as a substrate for Src family kinases, such as c-Src or Fyn, but not for Pyk2/RAFTK. Hyperosmotic stress induced phosphorylation of tyrosine 125 of alpha-synuclein via Pyk2/RAFTK, which acted through Src family kinases. Such phosphorylation was inhibited by PRAP expression, suggesting that PRAP negatively regulates alpha-synuclein phosphorylation following cell stress. In conclusion, PRAP functions as a downstream target for Pyk2/RAFTK and plays a role in alpha-synuclein phosphorylation.     

Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells

SHP-2, an SH2 domain-containing protein-tyrosine phosphatase, plays an important role in receptor tyrosine kinase-regulated cell proliferation and differentiation. Little is known about the activation mechanisms and the participation of SHP-2 in the activity of G protein-coupled receptors lacking intrinsic tyrosine kinase activity. We show that the activity of SHP-2 (but not SHP-1) is specifically stimulated by the selective alpha2A-adrenergic receptor agonist UK14304 and by lysophosphatidic acid (LPA) in Madin-Darby canine kidney (MDCK) cells. UK14304 and LPA promote the tyrosine phosphorylation of SHP-2 and its association with Grb2. The agonist-induced direct interaction of Grb2 with SHP-2 is mediated by the SH2 domain of Grb2 and the tyrosine phosphorylation of SHP-2. Rapid activation of Src family kinase by UK14304 preceded the SHP-2 activation. Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in MDCK cells, Fyn was the only one specifically associated with SHP-2, and the physical interaction between them, which requires the Src family kinase activity, was increased in response to the agonists. Pertussis toxin, PP1 (a selective Src family kinase inhibitor), or overexpression of a catalytically inactive mutant of Fyn blocked the UK14304- or LPA-stimulated activity of SHP-2, SHP-2 tyrosine phosphorylation, and SHP-2 association with Grb2. Therefore, we have demonstrated for the first time that the activation of SHP-2 by these Gi protein-coupled receptors requires Fyn kinase and that there is a specific physical interaction of Fyn kinase with SHP-2 in MDCK cells.     

Neuroligins determine synapse maturation and function

Synaptogenesis, the generation and maturation of functional synapses between nerve cells, is an essential step in the development of neuronal networks in the brain. It is thought to be triggered by members of the neuroligin family of postsynaptic cell adhesion proteins, which may form transsynaptic contacts with presynaptic alpha- and beta-neurexins and have been implicated in the etiology of autism. We show that deletion mutant mice lacking neuroligin expression die shortly after birth due to respiratory failure. This respiratory failure is a consequence of reduced GABAergic/glycinergic and glutamatergic synaptic transmission and network activity in brainstem centers that control respiration. However, the density of synaptic contacts is not altered in neuroligin-deficient brains and cultured neurons. Our data show that neuroligins are required for proper synapse maturation and brain function, but not for the initial formation of synaptic contacts.     

EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase

Ephrin kinases and their ephrin ligands transduce repulsion of cells in axon guidance, migration, invasiveness, and tumor growth, exerting a negative signaling on cell proliferation and adhesion. A key role of their kinase activity has been confirmed by mutant kinase inactive receptors that shift the cellular response from repulsion to adhesion. Our present study aimed to investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in ephrinA1/EphA2 signaling. LMW-PTP, by means of dephosphorylation of EphA2 kinase, negatively regulates the ephrinA1-mediated repulsive response, cell proliferation, cell adhesion and spreading, and the formation of retraction fibers, thereby confirming the relevance of the net level of tyrosine phosphorylation of Eph receptors. LMW-PTP interferes with ephrin-mediated mitogen-activated protein kinase signaling likely through inhibition of p120RasGAP binding to the activated EphA2 kinase, thereby confirming the key role of mitogen-activated protein kinase inhibition by ephrinA1 repulsive signaling. We conclude that LMW-PTP acts as a terminator of EphA2 signaling causing an efficient negative feedback loop on the biological response mediated by ephrinA1 and pointing on tyrosine phosphorylation as the main event orchestrating the repulsive response.     

Postsynaptic TrkC and presynaptic PTPσ function as a bidirectional excitatory synaptic organizing complex

Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express noncatalytic isoforms, suggesting additional functions. Here we discovered noncatalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans binding to axonal protein tyrosine phosphatase receptor PTPσ. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC-neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in?vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.     

N-cadherin and Neuroligins Cooperate to Regulate Synapse Formation in Hippocampal Cultures

Cadherins and neuroligins (NLs) represent two families of cell adhesion proteins that are essential for the establishment of synaptic connections in vitro; however, it remains unclear whether these proteins act in concert to regulate synapse density. Using a combination of overexpression and knockdown analyses in primary hippocampal neurons, we demonstrate that NL1 and N-cadherin promote the formation of glutamatergic synapses through a common functional pathway. Analysis of the spatial relationship between N-cadherin and NL1 indicates that in 14-day in vitro cultures, almost half of glutamatergic synapses are associated with both proteins, whereas only a subset of these synapses are associated with N-cadherin or NL1 alone. This suggests that NL1 and N-cadherin are spatially distributed in a manner that enables cooperation at synapses. In young cultures, N-cadherin clustering and its association with synaptic markers precede the clustering of NL1. Overexpression of N-cadherin at this time point enhances NL1 clustering and increases synapse density. Although N-cadherin is not sufficient to enhance NL1 clustering and synapse density in more mature cultures, knockdown of N-cadherin at later time points significantly attenuates the density of NL1 clusters and synapses. N-cadherin overexpression can partially rescue synapse loss in NL1 knockdown cells, possibly due to the ability of N-cadherin to recruit NL2 to glutamatergic synapses in these cells. We demonstrate that cadherins and NLs can act in concert to regulate synapse formation.     

Dual role for calcium in agrin signaling and acetylcholine receptor clustering

Agrin is a motoneuron‐derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin‐ and neuramindase‐induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR β‐subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin‐induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 69–79, 2002