Interaction and inhibitory cross-talk between endothelin and ErbB receptors in the adult heart

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 ET A such as G alpha(q), phospholipase C-beta 1, protein kinase C (PKC) epsilon, and PKC delta 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-1 beta (NRG1 beta) 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 1) prolonged exposure to ET-1; 2) degeneration of T-tubules; and 3) therapies targeted at erbB2 inhibition.