Regulated interaction of endothelin B receptor with caveolin-1.
The peptide hormone endothelin transmits various signals through G protein-coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR. ETBR formed a complex with caveolin-1 in cells in which these two proteins were coexpressed and in the mixture after purification and reconstitution (as examined by immunoprecipitation) suggesting the direct binding of ETBR with caveolin-1. The complex formed efficiently only when the ETBR was ligand-free or bound to an antagonist, RES-701-1, whereas the addition of ET-1 or another antagonist, BQ788, dissociated the complex, suggesting the structural recognition of ETBR by caveolin-1. In contrast, the ETAR bound to caveolin-1 regardless of ligand binding. Caveolin-1 utilized its scaffolding domain (residues 82-101) and the C-terminal domain (residues 136-178) to bind to ETBR, as for other signalling molecules. Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin-1 and decreased with the addition of ET-1. The disruption of caveolae by filipin reduced the ET-1-derived phosphorylation of ERK1/2. These results suggest the possibility that the binding to caveolin-1 retains the ligand-free ETBR in caveolae and regulates the ET signal.
Yamaguchi
Tomohiro
T
Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Japan.
Murata
Yasunobu
Y
Fujiyoshi
Yoshinori
Y
Doi
Tomoko
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D021941:Caveolae / Q000502:physiology; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D008168:Lung; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D014162:Transfection
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D017931:DNA Primers; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011994:Recombinant Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases