Calcium/calmodulin regulates signaling at the α_1A adrenoceptor

Cardiovascular functions are mediated by multiple 7-pass transmembrane receptors whose activation promotes contraction or relaxation of the tissues. The α₁ adrenoceptor type 1A plays important roles in the control of vascular tone and myocardial contractility via Ca²⁺-dependent actions. Here, using novel FRET-based biosensors, we identified a novel Ca²⁺-dependent interaction between calmodulin (CaM) and the human α_1A adrenoceptor at the juxtamembranous region of its 4th submembrane domain (SMD4_JM, a.a. 333-361). SMD4_JM houses the known nuclear localization signal of α_1A adrenoceptor (NLS, a.a. 334-349). We found that NLS itself also interacts with CaM, but with lower affinity and Ca²⁺ sensitivity, indicating that full interaction between CaM and α_1A receptor in this region requires segment a.a. 333-361. Combined K353Q/L356A substitutions in the non-NLS segment of SMD4_JM cause a 3.5-fold reduction in the affinity of CaM-SMD4_JM interaction. Overexpression of wild-type α_1A adrenoceptor in cells enhances phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) stimulated by A61603, while overexpression of the K353Q/L356A α_1A receptor mutant significantly reduces this signal. Norepinephrine stimulates intracellular Ca²⁺ signals that are higher in cells overexpressing wild-type receptor but lower in cells overexpressing the K353Q/L356A receptor compared to non-transfected cells in the same microscopic environments. These data support a novel and important role for Ca²⁺-dependent CaM interaction at SMD4_JM in α_1A adrenoceptor-mediated signaling.