The angiotensin II receptor type 1 (ATR) mediates many Ca-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca 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, a.a., 309-327). Simultaneous measurements of CaM binding and free Ca show that the interactions are Ca-dependent, with disparate K and EC50(Ca) values within the physiological range of cytoplasmic Ca. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215-242) and has an EC50(Ca) value in the range of resting cytoplasmic Ca, suggesting ATR-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 ATR inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant ATR with reduced CaM binding at each identified binding domain. AngII triggers transient Ca signals in cells expressing wild-type ATR. These signals are reduced in cells expressing mutant ATR with reduced CaM binding at SMD3 or SMD4, but are very slow-rising, low amplitude signal in cells expressing ATR with reduced CaM binding at SMD2. The data indicate that CaM interactions with ATR can occur at various domains, with different affinities, at different physiological Ca levels, and are important for ATR-mediated signaling.

The angiotensin II receptor type 1A (AT1R) is responsible for many effects of angiotensin II (AngII). Calmodulin (CaM) is essential for many cell functions due to its interactions with many proteins. We tested the idea that signaling via AT1R involves CaM at the receptor level via multiple binding domains. AT1R coimmunoprecipitates with CaM in vascular smooth muscle under resting conditions, association that is enhanced by stimulation with AngII or thapsigargin. To identify all CaM‐binding domains in AT1R and characterize their binding properties, we generated FRET biosensors in which each the 4 sub‐membrane domains (SMDs) in AT1R is flanked by enhanced variants of yellow and cyan fluorescence protein. We named these biosensors BSAT1Rx, with x denoting the amino acid numbering of the SMD sequence. In response to purified Ca2+‐saturated CaM, BSAT1R125‐141, BSAT1R215‐242 and BSAT1R309‐327, corresponding to SMD2, SMD3 and the juxta membranous segment SMD4 in human AT1R, display drastic responses that are characteristic of conformational changes caused by direct binding of CaM. BSAT1R53‐64, corresponding to SMD1, does not response to Ca2+‐CaM. Titrations of biosensor responses with purified CaM yielded Kd values of 44.7±1.00, 0.36± 0.05, and 0.51±0.01 microM, respectively for SMD2, 3, and the juxta‐membranous segment of SMD4. The respective EC50(Ca2+) values of these interactions were 4.1±0.11, 0.13 ± 0.006, and 1.26 ± 0.09 microM, determined by monitoring concurrent responses of the respective BSAT1Rx and a suitable Ca2+ indicator. The entire SMD3 is involved in interaction with CaM, as a truncated domain (a.a. 215‐232) displays substantial decreases in affinity and Ca2+ sensitivity. These data demonstrate that CaM is involved in AngII signaling via direct interactions with multiple domains in AT1R and allow prediction of interactions between CaM and individual SMDs under distinct physiological scenarios, including resting and stimulated conditions. Grant Funding Source: National Institutes of Health

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