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

Abstract

The angiotensin II type 1 receptor (AT 1 R) is a G␣ q/11 -coupled G protein-coupled receptor that is widely expressed in multiple tissues, including vascular smooth muscle cells, brain, and kidney. Activation of the AT 1 R 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 AT 1 R-induced alterations in membrane dynamics in human embryonic kidney 293 cells. We find that the activation of the AT 1 R 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. AT 1 R-stimulated membrane bleb formation is independent of RalA, RalB, Rac1, cdc42, Arf6, and Ras, but it involves RhoA. Furthermore, membrane blebbing activated by the AT 1 R 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 AT 1 R activation. The inhibition of the downstream RhoA effectors Rho kinase (ROCK) and myosin light chain kinase (MLCK) effectively attenuated AT 1 R-mediated membrane blebbing. Thus, we show that membrane blebbing in response to AT 1 R signaling is dependent on ␤-arrestin2 and is mediated by a RhoA/ ROCK/MLCK-dependent pathway.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways

“…Among the videos recorded upon cell activation by NKA, 30% display membrane blebbing of the cells. Activation of GPCRs has been described as triggering a cellular blebbing that lasts several minutes and is continuously dynamic (31). On the other hand, the blebbing observed during our screening occurred just after activation and was short-lasting.…”

Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes

“…The Rho/ROCK pathway is a key component of many membrane blebbing processes (37). We show that Rho/ROCK-dependent membrane blebbing is necessary for rapamycin-induced ACV release in articular chondrocytes.…”

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

“…UTP-induced migration is also reported to occur following the synthesis of osteopontin, via an ERK-and Rho-dependent mechanism (11). Interestingly, arrestins have been shown to modulate Rho-dependent membrane blebbing, an actin-dependent process known to contribute to cell motility (19). However, since this process is mediated by arrestin3 and inhibited by arrestin2, it appears unlikely to be the mechanism by which arrestin2 regulates agonist-driven migration in ASMC.…”