β-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells

Abstract

Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage ␤-arrestin, but not G q , to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of ␤-arrestin proteins, we overexpressed ␤-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, ␤-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to ␤-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration.

Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp

“…β-arrestins were found to promote clathrin-mediated internalization of GPCRs by interacting with, and scaffolding various components of, the clathrin-mediated endocytosis machinery. In particular, β-arrestin2 promotes the activation of ARF6 and endocytosis in the migration of vascular smooth muscle cells [56]. After overexpression in mouse embryonic fibroblasts, β-arrestin2 also enhanced low density lipoprotein receptor (LDLR) endocytosis (by 65%), through the interaction between β-arrestin2 with LDLR cytoplasmic tail [57].…”

Inhibition of 5‐Hydroxytryptamine Receptor 2B Reduced Vascular Restenosis and Mitigated the β‐Arrestin2–Mammalian Target of Rapamycin/p70S6K Pathway

“…Apart from canonical G‐protein–mediated signaling, G‐protein–coupled receptors also activate a β‐arrestin–dependent noncanonical pathway 40. β‐Arrestins have been reported to be involved in motility of SMCs 41. We found that silencing of β‐arrestin2 with siRNA transfection in SMCs (Figure S3A) markedly reduced the phosphorylation of mTOR and p70S6K induced by BW723C86 (Figure 5B).…”

“…40 b-Arrestins have been reported to be involved in motility of SMCs. 41 We found that silencing of b-arrestin2 with siRNA transfection in SMCs ( Figure S3A) markedly reduced the phosphorylation of mTOR and p70S6K induced by BW723C86 ( Figure 5B). These studies suggested that b-arrestin2 played a critical role in the 5-HT2BR-mediated activation of the mTOR/p70S6K pathway in SMCs.…”

“…It was reported that β‐arrestins can regulate myosin light chain phosphorylation to promote the motility of SMC 68. β‐Arrestin2 mediates angiotensin II–induced SMC migration in such a manner 41. More important, ergolines, such as lysergic acid diethylamide and ergotamine, displayed bias for β‐arrestin signaling at 5‐HT2BR 69.…”

“…68 b-Arrestin2 mediates angiotensin II-induced SMC migration in such a manner. 41 More important, ergolines, such as lysergic acid diethylamide and ergotamine, displayed bias for b-arrestin signaling at 5-HT2BR. 69 It is indicated that the b-arrestin-biased signalsome mediated the pathological effects of 5-HT2BR by converging the serotonergic signaling with the downstream mTOR/p70S6K pathways.…”