Inhibition of AT1 Receptor Internalization by Concanavalin A Blocks Angiotensin II-induced ERK Activation in Vascular Smooth Muscle Cells

Abstract

Angiotensin II (Ang II), 1 a major effector peptide of the renin-angiotensin system, is believed to play a critical role in the pathogenesis of cardiovascular remodeling associated with hypertension, heart failure, and atherosclerosis (1). Ang II binds to at least two types of receptor with high affinity, designated AT 1 and AT 2 receptors (2). We and others (3, 4) previously cloned the AT 1 receptor that belongs to the superfamily of heterotrimeric G protein-coupled receptors (GPCRs). The AT 1 receptor activates phospholipase C ␤ through the G q protein to generate inositol trisphosphate (IP 3 ) and diacylglycerol, which in turn releases calcium from intracellular stores and activates protein kinase C, respectively (5, 6). At present, the AT 1 receptor is thought to mediate almost all the known physiological and pathological effects of Ang II on the target cells such as vascular smooth muscle cells (VSMC) (7-9), cardiomyocytes (5), cardiac fibroblasts (10), and renal mesangial cells (11).Since the AT 1 receptor is a key mediator in the biologic mechanisms of the renin-angiotensin system, there has been considerable interest in defining its signaling pathways that mediate the cardiovascular physiology. In cultured VSMC, Ang II activates extracellular signal-regulated kinase (ERK), which is an obligatory step for the Ang II-induced cellular hypertrophy of VSMC (12). We and others recently reported that Ang II-induced calcium-dependent transactivation of the epidermal growth factor (EGF) receptor plays a predominant role in the ERK activation by Ang II in VSMC (13) and in cardiac fibroblasts (14). Src family kinase and Pyk2, a recently identified calcium-sensitive tyrosine kinase, seem to be involved in the EGF receptor transactivation by Ang II in VSMC (15).Recent studies of the ␤ 2 -adrenergic receptor suggest that agonist-promoted receptor internalization plays an important role in ERK activation (16). In Rat 1a fibroblasts, the GPCRmediated activation of ERK is sensitive to four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A (ConA), hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine (17). In addition, expression of dominant inhibitory mutants of ␤-arrestin1 or dynamin, which attenuate agonist-induced receptor internalization, inhibits ␤ 2 -adrenergic receptor-mediated ERK activation (16, 18). Since GPCRs appear to internalize through a clathrin-mediated process and AT 1 receptor internalization is blocked by ConA (19), we performed studies to examine the possible requirement of AT 1 receptor internalization for the receptor signaling using the receptor internalization blocker ConA and the receptor mutants with impaired internalization. We obtained evidence that AT 1 receptor internalization is not required for the early signaling events and the downstream ERK activation by the receptor in VSMC. Furthermore, we found that ConA blocked Ang II-induced activation of ERK through a distinct mechanism, the ConA-mediated proteolysis of the EGF recept...

Signal Transduction of Physiological Concentrations of Vasopressin in A7r5 Vascular Smooth Muscle Cells

“…Transactivation of EGFR has been implicated as a downstream mediator of PYK2 activation in angiotensin II-stimulated vascular smooth muscle cell hypertrophy (21,24,25,32). However, we found that direct activation of EGFR by binding of EGF did not stimulate Ca 2ϩ spiking despite activation of ERK1/2.…”

Calcium-sensing Receptor-mediated ERK1/2 Activation Requires Gαi2 Coupling and Dynamin-independent Receptor Internalization

“…3B, where the intracellular pool of CaR present at steady-state is no longer as prevalent, as well as in its ability to block EGF receptor-mediated ERK1/2 activation on the same blot (Fig. 3A), as the ability of concanavalin A to block the tyrosine kinase receptor-mediated activation of ERK1/2 has been demonstrated in some systems (33,36). These findings suggest that CaR internalization is partially required for ERK1/2 activation and further suggest the possibility that like other GPCRs, CaR activation of ERK1/2 by NPS R-467/ CaCl 2 and the later wave of 4 mM Ca 2ϩ activation may involve receptor cross-talk with the receptor-tyrosine kinase signaling pathway.…”

“…EGF receptor-mediated ERK1/2 activation (25 ng EGF/ml for 5 min) was used as a positive control to demonstrate that the concanavalin A was able to block this signaling pathway of this tyrosine kinase receptor pathway as previously demonstrated (33).…”

Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission

“…Interestingly, receptor internalization appears to be dierentially regulated since upon AR stimulation and EGFR transactivation the b 2 subtype is internalized together with the EGFR while the a 2A AR remains on the surface of transfected COS-7 cells . Remarkably, concanavalin A (ConA)-treatment of vascular smooth muscle cells blocked AT 1 R internalization, AngII-induced EGFR transactivation and MAP kinase activation demonstrating the necessity of an intact cytoskeleton for receptor downregulation and signal transmission (Tang et al, 2000).…”