Transmembrane helix 7 of the endothelin B receptor regulates downstream signaling.
Endothelin is a 21-amino acid peptide with a striking diversity of important biological responses, including, vasoconstriction, bronchoconstriction, and mitogenesis. Endothelin-1 binding to the endothelin B receptor (ETB), a member of the superfamily of G-protein-coupled receptors, was associated with catalytic activation of the extracellular-regulated kinase 2 (ERK2) and stimulation of AP-1 transcriptional reporter activity. A panel of single point mutations in transmembrane helix 6 (TM6), intracellular loop 3, and transmembrane helix 7 (TM7) were developed to study the structural requirements for ETB activation. Point mutations within highly conserved regions of TM6 and intracellular loop 3 were without effect on agonist-stimulated ERK activation. However, mutations within TM7 of the ETB significantly impacted ligand-stimulated downstream signaling. For example, nine point mutations within TM7 of the ETB were identified that prevented endothelin-stimulated ERK activation. Interestingly, the TM7 mutants fell into two classes; several exhibited greatly decreased AP-1 activity, relative to wild type ETB, whereas others displayed augmented endothelin-stimulated AP-1 transcriptional activity relative to wild type ETB. Our results suggest that TM7 of the ETB is involved in its activation mechanism and regulates agonist-stimulated ERK activation.
Vichi
P
P
Department of Biomedical Technologies, School of Allied Health, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA. pvichi@zoo.uvm.edu
Whelchel
A
A
Posada
J
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism; D004789:Enzyme Activation; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation
D019332:Endothelin-1; D008024:Ligands; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D018808:Transcription Factor AP-1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1