RESUMEN
The calcitonin gene-related peptide (CGRP) plays important roles as a neurotransmitter/neuromodulator in the central nervous system, and as a potent vasodilator when secreted from peripheral, perivascular nerves through its specific receptors. In this study, we cloned mouse cDNA counterparts of the human CGRP receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) and examined the signal transduction mechanism through the CGRP receptor. Mouse CRLR (mCRLR) is a 462-amino acid G protein-coupled heptahelical receptor, and mouse RAMP1 (mRAMP1) is a 148-amino acid single membrane-spanning protein with a short cytoplasmic portion. Specific binding of (125)I-CGRP was detected only when both mCRLR and mRAMP1 cDNAs were cotransfected to COS-7 cells, and the Kd value of the receptor was 2.2 x 10(-10) M. CGRP induced a marked elevation of the intracellular cAMP levels in COS-7 cells cotransfected with mCRLR and mRAMP1. CGRP signaling through the mCRLR/mRAMP1 receptor complex was found to increase the promoter activities of cyclic AMP responsive element and serum responsive element in the co-transfected HeLa cells. These results indicate that mCRLR and mRAMP1 constitute a functional mouse CGRP receptor for the transduction of CGRP signaling by PKA and extracellular signal-regulated kinase signal transduction pathways.
Asunto(s)
Proteínas de la Membrana/genética , Ratones/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Cultivadas/metabolismo , Chlorocebus aethiops , Clonación Molecular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , ADN Complementario/genética , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sustancias Macromoleculares , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Péptido Relacionado con el Gen de Calcitonina/química , Receptores de Péptido Relacionado con el Gen de Calcitonina/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Linfocitos T/metabolismo , TransfecciónRESUMEN
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with small platelets, severe eczema, and recurrent infections due to defects in the immune system. The disease arises from mutations in the gene encoding the WAS protein (WASP), which plays a role as an adaptor molecule in signal transduction accompanied by cytoskeletal rearrangement in T cells. To investigate the functional domain of WASP, we developed transgenic mice overexpressing the WASP N-terminal region (exon 1-5) including the Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain, in which the majority of mutations in WAS patients have been observed. WASP transgenic mice develop and grow normally under the specific pathogen-free environment, and showed normal lymphocyte development. However, proliferative responses and cytokine production induced by TCR stimulation were strongly inhibited in transgenic mice, whereas Ag receptor capping and actin polymerization were normal. These findings suggest that overexpressed Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain of WASP inhibits the signaling from TCR without coupling of cytoskeletal rearrangement. WASP transgenic mice shown here could be valuable tools for further understanding the WASP-mediated processes.