Received for publication June 7, 2006.
Revised February 28, 2007.
Accepted for publication March 1, 2007.

Interaction and Inhibitory Cross-Talk between Endothelin and ErbB Receptors in the Adult Heart

Abstract

Endothelin-1 (ET-1) regulates contractility and growth of the mammalian heart by binding ET_A and ET_B G-protein coupled receptors (GPCRs). To identify growth signaling pathways associated with ET-1 receptors in adult myocardium, a combined immunoprecipitation/proteomic analysis was performed. Signaling proteins thought to function downstream of ET_A such as Gq, PLC-1, PKC and PKC were identified in immunoprecipitates of ET_A by MALDI-TOF mass spectrometry. Also prominent were the growth factor receptor tyrosine kinases erbB2 and erbB4, as well as their downstream growth signaling effectors PI3-kinase, Akt, Raf-1, MEK and Erk. Western blot analysis confirmed co-immunoprecipitation of erbB2/4, PI3-kinase, and Akt with ET_A, and confocal microscopy revealed their co-localization in cardiac transverse tubules (T-tubules). The erbB4 receptor ligand neuregulin-1 (NRG1) 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 NRG1 mediated actions including erbB2/4 phosphorylation, serine phosphorylation of Akt, and negative inotropy. This inhibitory cross-talk between ET_A 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 ET_A and erbB pathways is fundamentally different in adult versus neonatal cardiac myocytes. The results may be relevant to cardiomyopathies associated with i) prolonged exposure to ET-1, ii) degeneration of T-tubules, and iii) therapies targeted at erbB2 inhibition.

Key words: Endothelin, NGF/EGF, Gq/11 family, Phospholipase C's, Protein Kinase C, Fluorescence techniques, Mass Spectroscopy

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