Changes in angiotensin II type 1 receptor signalling pathways evoked by a monoclonal antibody raised to the N-terminus.
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 microg/ml) inhibited both basal and angiotensin II (Ang II; 10(-7) mol/l)-stimulated [H(3)]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Calpha (PKCalpha) 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 PKCalpha, 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.
Xiao
Fang
F
School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK. f.xiao@ucl.ac.uk
Puddefoot
John R
JR
Barker
Stewart
S
Vinson
Gavin P
GP
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008297:Male; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000235:genetics
D000911:Antibodies, Monoclonal; D044140:Receptor, Angiotensin, Type 1; D018808:Transcription Factor AP-1; D000804:Angiotensin II; D051571:Protein Kinase C-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium