RESUMO
Synaptogenesis at the neuromuscular junction requires agrin-induced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MuSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.
Assuntos
Agrina/metabolismo , Citoesqueleto/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/metabolismo , Quinases da Família src/metabolismo , Agrina/farmacologia , Animais , Células COS , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Substâncias Macromoleculares , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Codorniz , Agregação de Receptores/fisiologia , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Transfecção , Vanadatos/farmacologia , Domínios de Homologia de src/fisiologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
The studies discussed in this review demonstrate that phosphorylation is an important mechanism for the regulation of ligand-gated ion channels. Structurally, ligand-gated ion channels are heteromeric proteins comprised of homologous subunits. For both the AChR and the GABA(A) receptor, each subunit has a large extracellular N-terminal domain, four transmembrane domains, a large intracellular loop between transmembrane domains M3 and M4, and an extracellular C-terminal domain (Fig. 1B). All the phosphorylation sites on these receptors have been mapped to the major intracellular loop between M3 and M4 (Table 1). In contrast, glutamate receptors appear to have a very large extracellular N-terminal domain, one membrane hairpin loop, three transmembrane domains, a large extracellular loop between transmembrane domains M3 and M4, and an intracellular C-terminal domain (Fig. 1C). Most phosphorylation sites on glutamate receptors have been shown to be on the intracellular C-terminal domain, although some have been suggested to be on the putative extracellular loop between M3 and M4 (Table 1). A variety of extracellular factors and intracellular signal transduction cascades are involved in regulating phosphorylation of these ligand-gated ion channels (Fig. 2). Once again, the AChR at the neuromuscular junction is the most fully understood system. Phosphorylation of the AChR by PKA is stimulated synaptically by the neuropeptide CGRP and in an autocrine fashion by adenosine released from the muscle in response to acetylcholine. In addition, acetylcholine, via calcium influx through the AChR, appears to activate calcium-dependent kinases including PKC to stimulate serine phosphorylation of the receptor. Presently, agrin is the only extracellular factor known to stimulate phosphorylation of the AChR on tyrosine residues. For glutamate receptors, non-NMDA receptor phosphorylation by PKA is stimulated by dopamine, while NMDA receptor phosphorylation by PKA and PKC can be induced via the activation of beta-adrenergic receptors, and metabotropic glutamate or opioid receptors, respectively. In addition, Ca2+ influx through the NMDA receptor has been shown to activate PKC. CaMKII, and calcineurin, resulting in phosphorylation of AMPA receptors (by CaMKII) and inactivation of NMDA receptors (at least in part through calcineurin). In contrast to the AChR and glutamate receptors, no information is presently available regarding the identities of the extracellular factors and intracellular signal transduction cascades that regulate phosphorylation of the GABA(A) receptor. Surely, future studies will be aimed at further clarifying the molecular mechanisms by which the central receptors are regulated. The presently understood functional effects of ligand-gated ion channel phosphorylation are diverse. At the neuromuscular junction, a regulation of the AChR desensitization rate by both serine and tyrosine phosphorylation has been demonstrated. In addition, tyrosine phosphorylation of the AChR or other synaptic components appears to play a role in AChR clustering during synaptogenesis. For the GABA(A) receptor, the data are complex. Both activation and inhibition of GABA(A) receptor currents as a result of PKA and PKC phosphorylation have been reported, while phosphorylation by PTK enhances function. The predominant effect of glutamate receptor phosphorylation by a variety of kinases is a potentiation of the peak current response. However, PKC also modulates clustering of NMDA receptors. This complexity in the regulation of ligand-gated ion channels by phosphorylation provides diverse mechanisms for mediating synaptic plasticity. In fact, accumulating evidence supports the involvement of protein phosphorylation and dephosphorylation of AMPA receptors in LTP and LTD respectively. There has been a dramatic increase in our understanding of the nature by which phosphorylation regulates ligand-gated ion channels. However, many questions remain unanswered. (AB
Assuntos
Canais Iônicos/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Fosforilação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de GABA-A/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Transdução de SinaisRESUMO
The microtubule protein Tctex-1 was cloned from Torpedo electroplax, a biochemical model of the neuromuscular junction, using the unique domain of Fyn in the yeast two hybrid system. Binding of Tctex-1 and Fyn also occurred in vitro. Torpedo Tctex-1 was contained within the molecular motor protein dynein. A Src class kinase was also complexed with dynein. Tctex-1 was enriched in electric organ vs. skeletal muscle, was present in the postsynaptic membrane, and coprecipitated with the acetylcholine receptor. The sequence of Tctex-1 contained a tyrosine phosphorylation motif and Tctex-1 could be phosphorylated by Fyn in vitro and in vivo. These data demonstrated that Tctex-1-containing dynein is a cytoskeletal element at the acetylcholine receptor-enriched postsynaptic membrane and suggested that Tctex-1 may be a substrate for Fyn.
Assuntos
Dineínas/metabolismo , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos , Proteínas Nucleares , Torpedo/genética , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dineínas/genética , Proteínas dos Microtúbulos/metabolismo , Dados de Sequência Molecular , Fosforilação , Torpedo/metabolismo , Região do Complexo-t do GenomaRESUMO
Most neurotransmitter receptors examined to date are either regulated by phosphorylation or contain consensus sequences for phosphorylation by protein kinases. The nicotinic acetylcholine receptor (AChR), which mediates depolarization at the neuromuscular junction, has served as a model for the study of the structure, function, and regulation of ligand-gated ion channels. The AChR is phosphorylated by protein kinase A, protein kinase C, and an unidentified protein tyrosine kinase. Tyrosine phosphorylation of the AChR is correlated with a modulation of the rate of receptor desensitization and is associated with AChR clustering. We showed that agrin, a neuronally derived extracellular matrix protein, induces AChR clustering and tyrosine phosphorylation. In addition, we identified two protein tyrosine kinases, Fyn and Fyk, that appear to be involved in the regulation of synaptic transmission at the neuromuscular junction by phosphorylating the AChR. The two kinases are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. As demonstrated by coimmunoprecipitation, Fyn and Fyk associate with the AChR. Furthermore, the AChR is phosphorylated in Fyn and Fyk immunoprecipitates. We investigated the molecular basis for the association of the AChR with Fyn and Fyk using fusion proteins derived from the kinases. The AChR bound specifically to the SH2 domain fusion proteins of Fyn and Fyk. The association of the AChR with the SH2 domains is dependent on the state of AChR tyrosine phosphorylation and is mediated by the delta subunit of the receptor. These data provide evidence that the protein tyrosine kinases Fyn and Fyk may act to phosphorylate the AChR in vivo.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores Nicotínicos/metabolismo , Tirosina/análogos & derivados , Agrina/farmacologia , Sequência de Aminoácidos , Animais , Galinhas , Camundongos , Dados de Sequência Molecular , Fosforilação , Fosfotirosina , Testes de Precipitina , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinapses/enzimologia , Torpedo , Tirosina/metabolismoRESUMO
The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine both in vitro and in vivo. To identify the protein tyrosine kinase that phosphorylates the receptor, we have previously cloned and characterized two protein tyrosine kinases, Fyn and Fyk, that are highly expressed in Torpedo electric organ, a tissue enriched in the AChR. Both kinases were shown by coimmunoprecipitation to be specifically associated with the AChR. In this study, we examined the molecular basis for the interaction of Fyn and Fyk with the AChR using fusion proteins containing the SH2 domains of the two kinases as affinity reagents. The AChR bound specifically and in a protein concentration-dependent manner to the SH2 domain fusion proteins of Fyn and Fyk. Quantitation of the association revealed that the binding of the AChR to Fyn and Fyk SH2 domain fusion proteins was to a single class of saturable high affinity sites. In addition, the association of the AChR with the SH2 domain fusion proteins was dependent on tyrosine phosphorylation of the AChR and was mediated by the delta subunit of the receptor. Furthermore, upon dissociation of the AChR into subunits, the delta subunit coimmunoprecipitated with both Fyn and Fyk. These data suggest that the association of the AChR with Fyn and Fyk is mediated by an interaction of the tyrosine-phosphorylated delta subunit of the receptor with the SH2 domains of the protein tyrosine kinases.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TorpedoRESUMO
The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine residues both in vitro and in vivo. This phosphorylation appears to regulate the rate of receptor desensitization and is associated with AChR clustering induced by the neuronal extracellular matrix protein agrin. To identify the protein tyrosine kinase(s) that phosphorylates the nicotinic receptor, we have used molecular cloning techniques to identify and characterize two protein tyrosine kinases that are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. One of the kinases was identified as the Torpedo homolog of neuronal fyn, whereas the other was a novel kinase we have named fyk due to its homology to both fyn and yes protein tyrosine kinases. Using antibodies to fyn and fyk, Fyn was shown to be a 55-kDa protein phosphorylated on tyrosine residues, whereas Fyk was a 56-kDa/53-kDa doublet phosphorylated on serine and tyrosine residues. At the mRNA and/or protein level, fyn and fyk were present in Torpedo electric organ, skeletal muscle, and brain. Both kinases were detected in the membrane fractions enriched in the AChR, with Fyn and Fyk representing 36 and 8%, respectively, of the protein tyrosine kinase activity in these postsynaptic membranes. In addition, Fyn and Fyk were shown by coimmunoprecipitation to be specifically associated with the AChR. Furthermore, the AChR was phosphorylated in Fyn and Fyk immunoprecipitates. These results indicate that Fyn and Fyk are involved in the regulation of postsynaptic membrane function and suggest that these protein tyrosine kinases may phosphorylate the AChR.
Assuntos
Órgão Elétrico/enzimologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Colinérgicos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Dados de Sequência Molecular , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Homologia de Sequência de Aminoácidos , Sinapses/metabolismo , Membranas Sinápticas/metabolismo , TorpedoRESUMO
Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.
Assuntos
Bombesina/farmacologia , Cálcio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Diglicerídeos/metabolismo , Sinergismo Farmacológico , Ácido Egtázico/farmacologia , Éteres/farmacologia , Fosfatos de Inositol/metabolismo , Secreção de Insulina , Ionomicina , Ionóforos/farmacologia , Potássio/farmacologia , Estimulação Química , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The amphibian tetradecapeptide, bombesin (BBS) has been shown to stimulate insulin secretion both in vivo and by pancreatic islet cells in vitro. To determine whether BBS can act directly on pancreatic beta cells, we examined its effects on insulin secretion by HIT-T15 cells (HIT cells), a clonal islet cell line. Addition of 100 nM BBS to HIT cells stimulated insulin release 25-fold within 30 sec. The rapid stimulatory effect of BBS on insulin release was short-lived: the secretory rate returned to basal levels after 90 min of BBS treatment. The decrease in the rate of insulin release in the continued presence of BBS was due not to depletion of intracellular insulin stores but to specific desensitization to this peptide. Stimulation of insulin secretion by BBS was dose dependent with an ED50 value (0.51 +/- 0.15 nM) similar to the concentration of BBS-like immunoreactive material in rat plasma. Five BBS analogs, including porcine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potencies of the analogs tested indicated that the COOH-terminal octapeptide sequence in BBS was sufficient for stimulation of release. In contrast, 14 peptides structurally unrelated to BBS did not alter insulin secretion. BBS action was synergistic with that of glucagon; insulin secretion in the presence of maximal concentrations of both peptides was greater than the additive effects of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 +/- 1% with an ID50 value of 3.2 +/- 0.3 nM. These results show that BBS stimulation of insulin secretion by a clonal pancreatic cell line closely parallels its effects in vivo and support the hypothesis that BBS stimulates insulin secretion by a direct effect on the pancreatic beta cell. The clonal HIT cell line provides a homogeneous cell preparation amenable for studies on the biochemical mechanisms of BBS action in the endocrine pancreas.