Changes in angiotensin II type 1 receptor signalling pathways evoked by a monoclonal antibody raised to the N-terminus

Abstract

The extracellular N-terminus of G-protein-coupled receptors may be involved in signalling events. We examined this in the angiotensin II type 1 receptor (AT1-R) using monoclonal antibody 6313/G2, raised against a conserved sequence in the N-terminal domain, and found it evokes inhibitory and stimulatory responses. In rat aortic smooth muscle cell (RASMC) primary cultures, 6313/G2 (2.5 μg/ml) inhibited both basal and angiotensin II (Ang II; 10−7 mol/l)-stimulated [H3]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Cα (PKCα) but gave a decrease in phosphorylated p44/42 extracellular signal-regulated kinases (ERK1/2) sustained from 10 min to 48 h compared with untreated control RASMC. In contrast, Ang II had no effect on PKCα, and, though it is acutely stimulatory (up to 5 min), it had no sustained effect on ERK1/2 either. Using Fura-2 and microfluorimetry, 6313/G2 added alone induced a transient increase in intracellular calcium ([Ca2+]i), with a characteristic response curve different from that of Ang II itself. The antibody was without effect on an Ang II-stimulated activator protein-1 reporter system, though it reduced unstimulated reporter activity. Such discriminatory effects on intracellular signalling suggest that the AT1-R N-terminus itself might be a target for therapeutic intervention in chronic vascular disease.