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 mg/ml) inhibited both basal and angiotensin II (Ang II; 10]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Ca (PKCa) 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 PKCa, 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 ([Ca 2C ] 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.

The renin–angiotensin system in the breast and breast cancer

“…Though not affecting angiotensin II binding to the receptor (Barker et al 1993) the antibody directly stimulates aldosterone secretion via the IP3 pathway in rat glomerulosa cells in vitro, though it also blocks PKC activation, apparently by interrupting receptor internalization (Kapas et al 1994. In other studies on rat vascular smooth muscle cells, basal and angiotensin-stimulated tritiated thymidine incorporation into rat arterial smooth muscle cells was inhibited by 6313/G2, inducing a transient increase in intracellular calcium in cultured rat arterial smooth muscle cells, but reducing PKC and MAPK signal transduction (Xiao et al 2008). A short-chain fragment variable of this antibody also blocked AT1 receptor-mediated caspase-3/7 inhibition in breast cancer cells and dose dependently gave significant tumour regression in breast cell xenografts in vivo.…”

Advances in the allostery of angiotensin II type 1 receptor

“…Uncertainty regarding the antigenic epitopes of the antibodies is a possible reason for the different effects of AT1R-AAs. It has been shown that unlike the agonistic effect of autoantibodies against ECL2, a monoclonal antibody against the N-terminal of AT1R, 6313/G2, inhibited AngII-induced cell proliferation [ 43 ]. Therefore, antibodies generated against different domains of a receptor can exert different modulating effects.…”