Novel regulations of the angiotensin II receptor type 1 by calmodulin

The angiotensin II receptor type 1 (AT₁R) 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 AT₁R-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 AT₁R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT₁R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca²⁺ signals in cells expressing wild-type AT₁R. These signals are reduced in cells expressing mutant AT₁R with reduced CaM binding at SMD3 or SMD4_JM, but are very slow-rising, low amplitude signal in cells expressing AT₁R with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT₁R can occur at various domains, with different affinities, at different physiological Ca²⁺ levels, and are important for AT₁R-mediated signaling.