Independent β-Arrestin2 and Gq/Protein Kinase Cζ Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Abstract

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and ␤-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the ␤-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated ␤-arrestinmediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of ␤-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase C. Moreover, ANG-and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. ␤-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and ␤-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.

Early Endosomal Antigen 1 (EEA1) Is an Obligate Scaffold for Angiotensin II-induced, PKC-α-dependent Akt Activation in Endosomes

“…EGFR-dependent outputs emanating from this domain activate at least two discrete signaling axes (6). One is represented by Erk1/2 and its downstream targets and another involves activation of p38 MAPK, PDK1, Akt, and p70S6K (7,8). This complexity of the AT1R signaling repertoire was anticipated by early findings in which we showed that Ang II stimulation of phospholipase-mediated generation of diacylglycerol is biphasic in VSMCs.…”

β-arrestin- but not G protein-mediated signaling by the “decoy” receptor CXCR7

“…Cells were split and used for assays 48 h after transfection. For rVSMCs, 80-90% confluent early passage (<3) VSMCs were transfected with siRNA using Lipofectamine 2000 transfection reagent (Invitrogen) according to the modified manufacturer's instructions (37). The siRNA sequence targeting β arrestin1 was 5-AGCCUUCUGUGCUGA-GAAC-3, corresponding to position 431-459 relative to the start codon.…”

Selectively Engaging β-Arrestins at the Angiotensin II Type 1 Receptor Reduces Blood Pressure and Increases Cardiac Performance

“…1 Sar,4 Ile, 8 Ile-angiotensin II (SII) induces ␤-arrestin recruitment, receptor internalization, and ␤-arrestin-mediated signals without activating G protein coupling (Wei et al, 2003;Ahn et al, 2004;Kim et al, 2009). Unfortunately, this peptide displays low receptor binding affinity (Holloway et al, 2002), which has made in vivo characterization of its effects challenging.…”