Abstract
Endothelin-1 (ET-1) regulates contractility and growth of the mammalian heart by binding endothelin receptor type A (ETA) and endothelin receptor type B (ETB) G-protein-coupled receptors. To identify growth signaling pathways associated with ET-1 receptors in adult myocardium, a combined immunoprecipitation/proteomic analysis was performed. Signaling proteins believed to function downstream of ETA such as Gαq, phospholipase C-β1, protein kinase C (PKC) ϵ, and PKCδ were identified in immunoprecipitates of ETA by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Also prominent were the growth factor receptor tyrosine kinases erbB2 and erbB4 and their downstream growth signaling effectors phosphoinositide-3 kinase (PI3 kinase), Akt, Raf-1, mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (Erk). Western blot analysis confirmed coimmunoprecipitation of erbB2/4, PI3 kinase, and Akt with ETA, and confocal microscopy revealed their colocalization in cardiac transverse tubules (T-tubules). The erbB4 receptor ligand neuregulin-1β (NRG1β) promoted erbB2/4 tryosine phosphorylation and Akt serine phosphorylation in ventricular myocytes, whereas treatment with ET-1 did not. This observation argues against ET-1 growth signaling occurring via erbB2/4 transactivation in adult myocardium. ET-1 did, however, stimulate Erk1/2 phosphorylation and substantially blunted several NRG1β-mediated actions, including erbB2/4 phosphorylation, serine phosphorylation of Akt, and negative inotropy. This inhibitory cross-talk between ETA and erbB2/4-Akt pathways was mimicked by a phorbol ester and blocked by pharmacological inhibition of PKC or MEK/Erk. The proteomic analysis and subsequent investigation of receptor cross-talk indicate that growth signaling between ETA and erbB pathways is fundamentally different in adult versus neonatal cardiac myocytes. The results may be relevant to cardiomyopathies associated with 1) prolonged exposure to ET-1; 2) degeneration of T-tubules; and 3) therapies targeted at erbB2 inhibition.
Footnotes
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This work was supported by National Institutes of Health grant HL081386 and by an award from the American Heart Association.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.027599.
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ABBREVIATIONS: ET-1, endothelin-1; ETA, endothelin receptor type A; ETB, endothelin receptor type B; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; NRG, neuregulin; PMA, phorbol 12-myristate-13-acetate; PLC, phospholipase C; PI3 kinase, phosphoinositide-3 kinase; PKC, protein kinase C; RTK, receptor tyrosine kinase; T-tubule, transverse tubule; Bim, bis-indoylmaleimide; IP, immunoprecipitate; IB, immunoblot; Erk1/2, extracellular signal-regulated kinases 1 and 2; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; MEK, mitogen-activated protein kinase kinase; NP-40, Nonidet P-40; PD98059, 2′-amino-3′-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; BQ788, N-cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine; BQ123, cyclo(d-Asp-Pro-d-Val-Leu-d-Trp.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received June 6, 2006.
- Accepted March 1, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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