The Angiotensin II Type 1 Receptor Induces Membrane Blebbing by Coupling to Rho A, Rho Kinase, and Myosin Light Chain Kinase

The angiotensin II type 1 receptor (AT1R) is a Gq/11-coupled G protein-coupled receptor that is widely expressed in multiple tissues, including vascular smooth muscle cells, brain, and kidney. Activation of the AT1R in vascular smooth muscle cells leads to alterations in actin-based membrane protrusions such as lamellipodia, filopodia, and membrane blebs that ultimately lead to cell migration, which is important for the regulation of vascular tone. In the present study, we examine the role of small G proteins in mediating AT1R-induced alterations in membrane dynamics in human embryonic kidney 293 cells. We find that the activation of the AT1R with 100 nM angiotensin II results in the rapid formation of membrane blebs at early time points of agonist stimulation that cease within 40 min of agonist stimulation. AT1R-stimulated membrane bleb formation is independent of RalA, RalB, Rac1, cdc42, Arf6, and Ras, but it involves RhoA. Furthermore, membrane blebbing activated by the AT1R is attenuated in the presence of the -arrestin aminoterminal domain, Ral GDP dissociation stimulator (RalGDS) -arrestin binding domain, and short interfering RNA (siRNA) depletion of -arrestin2. However, siRNA depletion of RalGDS protein did not affect membrane blebbing in response to AT1R activation. The inhibition of the downstream RhoA effectors Rho kinase (ROCK) and myosin light chain kinase (MLCK) effectively attenuated AT1R-mediated membrane blebbing. Thus, we show that membrane blebbing in response to AT1R signaling is dependent on -arrestin2 and is mediated by a RhoA/ ROCK/MLCK-dependent pathway. Angiotensin II (AngII) is an octapeptide hormone and the active component of the renin-angiotensin system. It regulates blood pressure and volume, thirst, and sympathetic nervous activity, and it has a role in vascular remodeling in hypertension (Touyz and Schiffrin, 2000). The majority of the physiological effects of AngII are mediated through the angiotensin II type 1 receptor (AT1R), which is predominantly localized to vascular smooth muscle cells in the vasculature. AT1R-mediated intracellular signaling cascades transduce vascular effects such as contraction, cell growth, migration,