Transmembrane Helix 7 of the Endothelin B Receptor Regulates Downstream Signaling

Abstract

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. Endothelin-1 (ET)1 is a 21-amino acid peptide that binds to a G-protein-coupled receptor (GPCR), of which there are two subtypes: endothelin receptor A, and endothelin receptor B (ETB). Receptor binding triggers a diverse spectrum of physiological effects including vasoconstriction, mitogenesis, and embryonic development (1-5).Consistent with the complex biology of endothelin, binding of the peptide to its receptor initiates several important cellular signaling pathways, including increases in cytosolic free calcium, activation of the Src cytosolic tyrosine kinase, tyrosine phosphorylation of the Shc adapter protein, ERK, and c-Jun NH 2 -terminal kinase mitogen-activated protein kinase activation, protein kinase C involvement, activation of phosphatidylinositol 3-kinase, and stimulation of the epidermal growth factor receptor tyrosine kinase activity (3, 6, 7, 9 -14).The endothelin receptors are members of a superfamily of G-protein-coupled receptors that are thought to activate downstream signaling networks through their dynamic interaction with heterotrimeric G-proteins. Mutational studies of the endothelin receptors have defined extracellular loop 2 as being critical for ligand contact and binding, whereas the cytoplasmic tail has been implicated as important for coupling to G-proteins (15-17).The relationship between ligand binding affinity and receptor-G-protein interaction is described by the ternary complex model of receptor activation (18). It is widely thought that a small fraction of receptors reside in the activated conformation and that agonist binding stabilizes this conformation, which results in activation of the associated G-protein. The dynamic interaction ...

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB

“…In contrast, substitution Tyr129His in ET A resulted in an increased affinity of ET‐3 and Sfx6c, although still 10‐fold to 10 000‐fold less active than in ET B 27–29. Ligand binding sensitive mutations are reported for charged residues in TMH2 (Asp2.50, Asp2.57, Lys2.64) and TMH7 (Asp7.35)30–34. However, data on these mutants do not explain the high selectivity in the binding of peptide ligands in endothelin receptor subtypes.…”

“…The molecular basis explaining ligand selectivity in ET A and ET B is poorly understood. In addition, the apparent binding affinities of peptide ligands such as ET‐1 (0.01–0.06 nM) are remarkably an order of magnitude higher than those of small molecules such as Bosentan (4.7 nM)34–37. A detailed molecular description of peptide ligand recognition, binding and initial receptor activation has, therefore, a vital importance in understanding the molecular mechanisms in endothelin receptor action as well as in the design of new ligands with binding affinities comparable to ET‐1.…”

The TXP Motif in the Second Transmembrane Helix of CCR5

“…Mutations of conserved prolines in other helices, notably TM5 (32) and TM7 (33,34), were also found to cause significant perturbations. For instance, proline mutations in the conserved NPXXY motif in TM7 often produce particularly strong phenotypes, including impaired activity (33,35,36).…”

Functional Characterization of Three Mutations of the Endothelin B Receptor Gene in Patients With Hirschsprung’s Disease: Evidence for Selective Loss of Gi Coupling

“…Vichi et al (36) have shown recently that some TMD7 mutants of the rat ET B receptor have normal ERK activation by ET-1 but a reduced AP-1 transcriptional activation. We tested the activation of AP-1 transcription by the mutant G57S and R319W hET B receptors by transient transfection of an AP-1/ luciferase reporter construct.…”