Biosensor‐based identification and characterization of three distinct calmodulin‐binding domains in the human angiotensin II receptor type 1A (LB569)

Abstract

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