ABSTRACT
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.
Subject(s)
Membrane Proteins/genetics , Mice/genetics , Receptors, Calcitonin Gene-Related Peptide/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells/metabolism , Calcitonin Gene-Related Peptide/pharmacology , Cells, Cultured/metabolism , Chlorocebus aethiops , Cloning, Molecular , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , DNA, Complementary/genetics , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System/drug effects , Macromolecular Substances , Mice, Inbred C57BL , Molecular Sequence Data , Organ Specificity , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Proteins , Receptors, Calcitonin Gene-Related Peptide/chemistry , Receptors, Calcitonin Gene-Related Peptide/drug effects , Recombinant Fusion Proteins/metabolism , Second Messenger Systems/drug effects , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , T-Lymphocytes/metabolism , TransfectionABSTRACT
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.
