Abstract 1222: Direct Evidence for Role of Caveolin1 and Microtubules/Actin Cytoskeleton in AT1 Receptor Trafficking and Signaling in Living Cells: Real-time Imaging Analysis
Angiotensin II (Ang II) promotes growth primarily through the Ang II type 1 receptor (AT1R). We showed using biochemical cell fractionation that AT1R movement from heavy membrane fractions to caveolin1 (Cav1)-enriched lipid rafts membranes is required for transactivation of EGF receptor (EGF-R) and requires intact microtubule(MT)/actin cytoskeleton as well as Cav1. There is little information concerning the real-time movement of AT1R during Ang II stimulation in living cells. Here we applied live cell imaging of GFP-tagged AT1R stably transfected in CHO cells and transiently transfected in VSMC using spinning disc confocal microscopy. We found that GFP-AT1R accumulated at discrete membrane areas within 1–5 min of Ang II stimulation. First, GFP-AT1R showed rapid lateral movement in vescicle-like structures to form plasma membrane patches which may be the posited specialized signaling domains within 1 min (2.0-fold increase). Second, Ang II caused dynamic formation of membrane ruffles where GFP-AT1R concentrated within 2 min (3.0-fold increase). Third, GFP-AT1R moved into filopodia-like structures and perinuclear compartment within 5 min (2.5-fold increase). Of note, these remarkedly dynamic, organized movements of AT1R were abolished by depolymerization of MTs with nocodazole or disruption of actin cytoskeleton with cytochalasin D, which block Ang II-induced EGF-R transactivation (75% and 81%, respectively) and its downstream Akt phosphorylation (76% and 79%, respectively). Moreover, disrupting caveolae/lipid rafts with β-cyclodextrin disorganized the direction of AT1R movement and Cav1 siRNA completely inhibited the formation of membrane ruffles, dynamic concentration of GFP-AT1R into these structures and lateral movement as well as Akt phosphorylation (92% inhibition). In summary, these live cell imaging data support our original biochemical findings that Ang II promotes AT1R movement within the plasma membrane in a Cav1- and MT/actin cytoskeleton-dependent manner, to form new signaling domains which are important for full expression of the Ang II signaling repertoire. These findings also provide new evidence that ruffle-like structures may represent previously unsuspected signaling domains.