Endothelin is a small peptide that is a potent bronchoconstrictor, mitogen for airway smooth muscle (ASM), and is believed to be involved in the pathogenesis of asthma. To understand how endothelin stimulates the proliferation of ASM cells in culture, we evaluated the relationship between mitogen activated protein (MAP) kinase activation and cell proliferation. Endothelin is a potent stimulator of the extracellular regulated kinase 2 (ERK2) subgroup of MAP kinases, and ERK2 activation was tightly correlated with the proliferation of rat ASM cells. PD98059, a small molecule inhibitor of MEK (MAP or ERK kinase) was used to establish the role of ERK2 activation in the endothelin-stimulated signal transduction pathway leading to cell proliferation. While PD98059 significantly inhibited the ability of endothelin to activate ERK, the drug did not appear to effect the catalytic activity of an activated MEK mutant, or ERK in vitro. The data suggest that the mechanism of PD98059 inhibition of the ERK2 pathway in ASM cells may involve inhibition of MEK activation. The endothelin signal transduction pathway that culminates in ERK2 activation was dependent on protein kinase C (PKC), since depletion of PKC significantly inhibited the ability of endothelin to activate ERK2. Taken together, the data imply that activation of ERK is a critical endpoint in the endothelin signal transduction pathway since inhibition of this kinase inhibits endothelin-induced ASM cell proliferation.
Endothelin is a 21-amino acid peptide with remarkably diverse biological properties, including potent vasoconstriction, induction of mitogenesis, and a role in the development of blood vessels. In the present study, stimulation of the endothelin B receptor was found to activate three distinct mitogen-activated protein kinase signal transduction pathways, the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK), and p38 kinase. These mitogen-activated protein kinase isozymes are thought to mediate very different biological outcomes, suggesting that the observed pattern of kinases activation may be important for the diverse biological properties of endothelin. The cytoplasmic tail of the endothelin B receptor was found to be required for activation of all three mitogen-activated protein kinases and stimulation of intracellular calcium levels. An endothelin B receptor truncated at the C-terminal tail was not able to stimulate the mitogen-activated protein kinases or increase cytosolic free calcium. Furthermore, ectopic expression of the cytoplasmic tail attenuated signaling through the wild type receptor. The observed ERK activation appeared to be mediated by heterotrimeric G proteins, since ectopic expression of a transducin ␣-subunit inhibited endothelin-stimulated ERK activation. The data suggest that the cytosolic tail of the endothelin B receptor is involved in calcium mobilization and mitogen-activated protein kinase activation via a G protein-dependent mechanism. Endothelin (ET)1 is a 21-amino acid peptide with diverse physiological effects on cellular development, differentiation, vasoconstriction, and mitogenesis. There are three endothelin isoforms, ET-1, ET-2, and ET-3, that have preference for different endothelin receptor subtypes. The endothelin A (ETA) receptor binds ET-1, ET-2, and ET-3 with decreasing affinities, whereas the endothelin B (ETB) receptor binds ET-1, ET-2, and ET-3 with similar affinity; both are members of the G proteincoupled receptor (GPCR) superfamily (1-7).The putative ET receptor topology includes three extracellular domains, three intracellular cytosolic loops, and a cytoplasmic C-terminal tail, separated by seven highly hydrophobic regions thought to span the lipid bilayer. Ligand binding and specificity is conferred by the conformation of the extracellular portions of the receptor, whereas the intracellular domains physically interact with heterotrimeric G proteins to initiate signal transduction events. Receptor-G protein complexes are thought to form the high affinity ligand binding state of the receptor (8). For example, constitutively activated adrenergic and muscarinic receptor mutants have increased affinity for agonist with little effect on antagonist binding, supporting the view that the receptor-G protein complex is the high affinity binding complex (9, 10).Endothelin receptor mutagenesis studies suggest that residues within extracellular loop two are important for ligand binding. The intracellular domains of the receptor, thought to play a role in...
Endothelin (ET)-1 is a 21-amino-acid peptide that is a potent vasoconstrictor and mitogen. By binding to its G-protein coupled receptor, ET-1 stimulates the proliferation of airway smooth-muscle (ASM) cells, which may be involved in the pathogenesis of asthma. The ETB receptor stimulates activation of the extracellular regulated kinase 2 (ERK2), which is thought to be required for proliferation of ASM cells. Our findings reveal that ET rapidly activates Raf, and that dominant-negative Raf interferes with ET-induced ERK activation in ASM cells. Expression of the amino-terminal Ras-binding domain of Raf inhibited ET-induced ERK activation, suggesting that ET-stimulated Raf activation is a Ras-dependent process. Furthermore, ET-stimulated ERK and Raf activation in ASM cells require calcium influx; chelating extracellular calcium or preventing calcium influx through calcium channels inhibited ET-stimulated, but not phorbol ester-stimulated, ERK and Raf activation.
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 ...
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