Determination of the Exact Molecular Requirements for Type 1 Angiotensin Receptor Epidermal Growth Factor Receptor Transactivation and Cardiomyocyte Hypertrophy

Abstract

Abstract-Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and ␤-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [ 32 P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the ␤-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a ␤-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or ␤-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor. A ngiotensin II (Ang II) contributes to progression of left ventricular hypertrophy, a major independent risk factor for myocardial infarction and sudden death, 1 yet the molecular mechanisms governing cardiac hypertrophy remain controversial. Recently, a new paradigm for Ang II hypertrophy has emerged. We and others have shown that angiotensin type 1 receptors (AT 1 Rs) hijack the epidermal growth factor (EFR) receptor (EGFR) via a process termed the "triple membrane-passing signaling paradigm of EGFR transactivation." 2-5 More specifically, AT 1 R activation causes metalloprotease-dependent EGF-like ligand release and subsequent EGFR binding to initiate hypertrophic signaling cascades. Yet the process is likely more complicated: multiple G protein-coupled receptor (GPCR) signals, a disintegrin and metalloproteases (ADAMs), EGF-like ligands, and EGFR subtypes have been implicated in this process. 2 A major focus of Ang II research is the mechanism by which the AT 1 R signals to the EGFR. Recent studies suggested a G protein-independent pathway for growth activation, 6 -9 despite evidence that G␣ q/11 , a heterotrimeric G protein that couples to the AT 1 R, is essential for hypertrophy. 10 Indeed, others indicate that G-protein coupling to AT 1 R is required for EGFR transactivation in COS-7 and vascular smoot...

A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

“…3B). Robust knockdown of G q/11 (GNAQ), essential for AT 1 R-mediated cardiomyocyte hypertrophy (Smith et al, 2011) and the development of cardiac hypertrophy in mice (Wettschureck et al, 2001), also abolished EGFR and ERK1/2 phosphorylation following AngII stimulation (Fig. 3C,D).…”

“…Since then, we and others have demonstrated that the AT 1 R can 'hijack' EGFR family signalling machinery and this is crucial for the downstream AT 1 Rmediated growth signalling and functional effects, such as cellular hypertrophy and/or proliferation (Asakura et al, 2002;Eguchi et al, 2001;Mifune et al, 2005;Thomas et al, 2002). Various mechanisms have been described to explain AT 1 R-EGFR transactivation, including metalloproteinase-mediated EGF ligand shedding (Mifune et al, 2005;Ohtsu et al, 2006), Ca 2+ -mediated Pyk2 activation (Eguchi et al, 1999), or direct interaction of the EGFR with the activated AT 1 R, through its tyrosine 319 residue (Seta and Sadoshima, 2003), although this does not appear to be the mechanism in a rat cardiomyocyte model (Smith et al, 2011).…”

Investigations into the role of the ErbB4 receptor in cardiomyocyte hypertrophy and adult cardiac function

“…Thus, it is possible that previous studies using AG1478 at 5 μM may have attributed actions to ErbB1 activation that are in fact being mediated by ErbB4 or other tyrosine kinases. Indeed, we have previously shown that 5 μM AG1478 is required to abolish Ang II-induced ERK1/2 activation and cardiomyocyte hypertrophy (Thomas et al, 2002;Smith et al, 2011), which suggests that multiple ErbB receptor subtypes may involved in this process.…”

“…In hepatic or breast cancer cell lines, PKC mediates the release of EGF-like factors, contributing to transactivation (Shah et al, 2002;Muscella et al, 2003). In cardiomyocytes and vascular smooth muscle cells, it has been shown that Ca 2+ but not PKC is essential for ErbB1 transactivation (Eguchi et al, 1998;Thomas et al, 2002;Smith et al, 2011). (Asakura et al, 2002;Saito et al, 2002;Mifune et al, 2005;Ohtsu et al, 2006b).…”

“…Whilst Ang II can cause cardiac hypertrophy subsequent to hypertension, it can also directly induce cardiomyocyte hypertrophy (Paradis et al, 2000;Schultz et al, 2002). We have previously shown that Ang II can directly promote cardiomyocyte hypertrophy via activation of the AT 1A R and Gq protein coupling (Thomas et al, 2002;Smith et al, 2011). At the molecular level, Ang II-induced cardiomyocyte hypertrophy is characterized by the re-induction of the 'fetal gene program' (increased expression of ANP, CyclinD and MLC-2V), along with reorganization of the cardiomyocyte sarcomere (Sadoshima et al, 1993;Aoki et al, 1998).…”