Novel regulations of the angiotensin II receptor type 1 by calmodulin

The angiotensin II receptor type 1 (AT(1)R) mediates many Ca2+-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca2+ signals in cells. Two locations on the receptor's submembrane domains (SMD) 3 and 4 are known to interact with CaM. However, the binding sites for CaM, biochemical properties of the interactions, and their functional impact are not fully understood. Using a FRET-based screening method, we identified a new binding site for CaM on SMD2 (a.a. 125-141), in addition to SMD3 and the juxtamembranous region of SMD4 (SMD4(JM), a.a., 309-327). Simultaneous measurements of CaM binding and free Ca2+ show that the interactions are Ca2+-dependent, with disparate K-d and EC50(Ca2+) values within the physiological range of cytoplasmic Ca2+. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215-242) and has an EC50(Ca2+) value in the range of resting cytoplasmic Ca2+, suggesting AT(1)R-CaM interaction can occur in resting conditions in cells. AngII induces robust ERK1/2 phosphorylation in primary vascular smooth muscle cells. This effect is suppressed by AT(1)R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT(1)R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca2+ signals in cells expressing wild-type AT(1)R. These signals are reduced in cells expressing mutant AT(1)R with reduced CaM binding at SMD3 or SMD4(JM), but are very slow-rising, low amplitude signal in cells expressing AT(1)R. with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT(1)R can occur at various domains, with different affinities, at different physiological Ca2+ levels, and are important for AT(1)R-mediated signaling.