The Small GTPase Ral Couples the Angiotensin II Type 1 Receptor to the Activation of Phospholipase C- 1

The angiotensin II type 1 receptor (AT1R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT1R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) via its association with G q/11, leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT1R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT1R, but that RalA expression is required for AT1R-stimulated IP formation but not 5-HT2A receptor-mediated IP formation. AT1R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the -arrestin-dependent plasma membrane translocation of RalGDS. G q/11 small interfering RNA (siRNA) treatment also significantly attenuates both AT1Rand 5-HT2A receptor-stimulated IP formation after 30 min of agonist stimulation. PLC1 has been reported to be activated by RalA, and we show that AT1R-stimulated IP formation is attenuated after PLC1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC1 stimulation. The angiotensin II type 1 receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) superfamily and regulates cellular function in a variety of tissues (de Gasparo et al., 2000). In the vasculature, angiotensin II (AngII)-mediated stimulation of the AT1R activates a myriad of cell signaling cascades that regulate vascular smooth muscle cell tone, growth, apoptosis, migration, and extracellular matrix deposition (Ushio-Fukai et al., 1998; Touyz and Schiffrin, 2000; Haendeler et al., 2003). The AT1R activates the G q/11 class of heterotrimeric G proteins that stimulate phospholipase C, leading to diacylglycerol and inositol-1,4,5-trisphosphate (IP3) formation, the release of Ca 2 from intracellular stores, and subsequent activation of protein kinase C. There are six families of phospholipase C isozymes (PLC, PLC, PLC, PLC, PLC, and PLC) that generate diacylglycerol and IP3 from phosphatidylinositol 4, 5-bisphosphate (PIP2) (Suh et al., 2008). The AT1R has traditionally been demonstrated to activate PLCbut can also induce tyrosine phosphorylation of PLC1 leading to the production of IP3 (Lea et al., 2002; Ochocka and Pawelczyk, 2003; Suh et al.,