RESUMO
Muscarinic activation of tracheal smooth muscle (TSM) involves a M(3)AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Galpha(i/o) subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Galpha(q16), whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Galpha(q15/16) stimulated the NPR-GC. Coupling of alpha(q16) to M(3)AChR is supported by MAS decreased [(3)H]QNB binding, being abolished after M(3)AChR-4-DAMP-alkylation. Anti-i(3)M(3)AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i(3)M(3)AChR (M(3)P) was more potent than MAS increasing GTPgamma [(35)S] and decreasing the [(3)H]QNB activities. Coupling between NPR-GC and Galpha(q16) was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Galpha(q16), also showing an immunoreactive heterotrimeric-G-beta-subunit. These data support the existence of a novel transducing cascade, involving Galpha(q16)beta gamma coupling M(3)AChR to NPR-GC.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Guanilato Ciclase/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Western Blotting , Bovinos , Cromatografia de Afinidade , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Guanosina Trifosfato/farmacologia , Guanilato Ciclase/isolamento & purificação , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Solubilidade/efeitos dos fármacos , Tripsina/metabolismo , Venenos de Vespas/química , Venenos de Vespas/farmacologiaRESUMO
The integrity of the Ras/Raf/mitogen-activated protein kinase (MAPK) cascade is critical for maintenance of T cell tolerance, a process that fails in patients with systemic lupus erythematosus (SLE). In this study we have examined the activity of mitogen-activated protein kinases ERK-1 and ERK-2 in resting and TCR-activated peripheral blood T lymphocytes from patients with SLE. We also examined the binding of Ras guanine nucleotide exchange factor, human Son of Sevenless (hSos), to cytosolic adapter protein growth factor receptor-bound protein 2. T cells from lupus patients showed diminished catalytic activity and TCR-driven dual phosphorylation of ERK-1 and ERK-2 upon stimulation through the TCR/CD3 receptor, a defect that may be related to altered translocation of hSos to the Ras/Raf membrane complex and diminished nuclear translocation of trans-acting factor AP-1. Defective MAPK activity triggered by TCR/ CD3 activation may alter the coordination of signals needed for normal interleukin-2 production and maintenance of tolerance in lupus T cells.