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1.
J Biol Chem ; 289(1): 203-14, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24240098

RESUMO

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-ß and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-ß and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, ßK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined ßK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the ß and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.


Assuntos
Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Receptores Nicotínicos/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/genética , Complexo de Golgi/genética , Humanos , Camundongos , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores Nicotínicos/genética
2.
J Neuroimmunol ; 373: 577978, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36240543

RESUMO

Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a neuromuscular autoimmune disease belonging to a growing group of IgG4 autoimmune diseases (IgG4-AIDs), in which the majority of pathogenic autoantibodies are of the IgG4 subclass. The more prevalent form of MG with acetylcholine receptor (AChR) antibodies is caused by IgG1-3 autoantibodies. A dominant role for IgG4 in autoimmune disease is intriguing due to its anti-inflammatory characteristics. It is unclear why MuSK autoantibodies are predominantly IgG4. We hypothesized that MuSK MG patients have a general predisposition to generate IgG4 responses, therefore resulting in high levels of circulating IgG4. To investigate this, we quantified serum Ig isotypes and IgG subclasses using nephelometric and turbidimetric assays in MuSK MG and AChR MG patients not under influence of immunosuppressive treatment. Absolute serum IgG1 was increased in both MuSK and AChR MG patients compared to healthy donors. In addition, only MuSK MG patients on average had significantly increased and enriched serum IgG4. Although more MuSK MG patients had elevated serum IgG4, for most the IgG4 serum levels fell within the normal range. Correlation analyses suggest MuSK-specific antibodies do not solely explain the variation in IgG4 levels. In conclusion, although serum IgG4 levels are slightly increased, the levels do not support ubiquitous IgG4 responses in MuSK MG patients as the underlying cause of dominant IgG4 MuSK antibodies.


Assuntos
Imunoglobulina G , Miastenia Gravis , Humanos , Autoanticorpos
3.
Front Immunol ; 11: 707, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32457737

RESUMO

Thirty to fifty percent of patients with acetylcholine receptor (AChR) antibody (Ab)-negative myasthenia gravis (MG) have Abs to muscle specific kinase (MuSK) and are referred to as having MuSK-MG. MuSK is a 100 kD single-pass post-synaptic transmembrane receptor tyrosine kinase crucial to the development and maintenance of the neuromuscular junction. The Abs in MuSK-MG are predominantly of the IgG4 immunoglobulin subclass. MuSK-MG differs from AChR-MG, in exhibiting more focal muscle involvement, including neck, shoulder, facial and bulbar-innervated muscles, as well as wasting of the involved muscles. MuSK-MG is highly associated with the HLA DR14-DQ5 haplotype and occurs predominantly in females with onset in the fourth decade of life. Some of the standard treatments of AChR-MG have been found to have limited effectiveness in MuSK-MG, including thymectomy and cholinesterase inhibitors. Therefore, current treatment involves immunosuppression, primarily by corticosteroids. In addition, patients respond especially well to B cell depletion agents, e.g., rituximab, with long-term remissions. Future treatments will likely derive from the ongoing analysis of the pathogenic mechanisms underlying this disease, including histologic and physiologic studies of the neuromuscular junction in patients as well as information derived from the development and study of animal models of the disease.


Assuntos
Músculos/patologia , Miastenia Gravis/enzimologia , Miastenia Gravis/patologia , Corticosteroides/uso terapêutico , Animais , Feminino , Subtipos Sorológicos de HLA-DR/genética , Haplótipos , Humanos , Imunoglobulina G/imunologia , Camundongos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Colinérgicos/genética
4.
J Neurosci ; 28(45): 11468-76, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-18987183

RESUMO

At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor that mediates agrin-induced clustering. Using chimeric proteins in which CD4 is fused to the large intracellular loop of each of the AChR subunits we found that agrin induced clustering of only chimeras containing the beta subunit loop. By making deletions in the beta loop we defined a 20 amino-acid sequence that is sufficient for clustering. The sequence contains a conserved tyrosine (Y390) whose phosphorylation is induced by agrin and whose mutation abolished clustering of beta loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChR beta subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect of betaY390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4-beta loop chimeras in a phosphorylation-dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR-beta(3F/3F), which lacks beta loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the beta subunit motif increases the stoichiometry of rapsyn binding to the AChR, thereby helping to stably cluster the receptor and anchor it at high density in the postsynaptic membrane.


Assuntos
Agrina/farmacologia , Células Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Receptores Nicotínicos/metabolismo , Motivos de Aminoácidos/fisiologia , Análise de Variância , Animais , Bungarotoxinas/metabolismo , Linhagem Celular Transformada , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoprecipitação , Camundongos , Modelos Moleculares , Células Musculares/fisiologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Transfecção/métodos , Tirosina/metabolismo
5.
J Neurobiol ; 50(1): 69-79, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11748634

RESUMO

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 beta-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.


Assuntos
Agrina/fisiologia , Cálcio/fisiologia , Receptores Colinérgicos/fisiologia , Animais , Cálcio/farmacologia , Células Cultivadas , Dimerização , Ativação Enzimática , Espaço Extracelular/fisiologia , Camundongos , Neurônios Motores/fisiologia , Neuraminidase/farmacologia , Junção Neuromuscular/fisiologia , Fosforilação , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Sinapses/fisiologia , Transfecção
6.
J Biol Chem ; 278(9): 7350-9, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12486121

RESUMO

The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsyn-AChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.


Assuntos
Agrina/metabolismo , Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Células COS , Linhagem Celular , Relação Dose-Resposta a Droga , Immunoblotting , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Músculos/citologia , Ligação Proteica , Ratos , Ratos Wistar , Fatores de Tempo
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