Calcium/calmodulin regulates signaling at the α1A 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 α1A adrenoceptor at the juxtamembranous region of its 4th submembrane domain (SMD4JM, a.a. 333-361). SMD4JM houses the known nuclear localization signal of α1A 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 α1A receptor in this region requires segment a.a. 333-361. Combined K353Q/L356A substitutions in the non-NLS segment of SMD4JM cause a 3.5-fold reduction in the affinity of CaM-SMD4JM interaction. Overexpression of wild-type α1A adrenoceptor in cells enhances phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) stimulated by A61603, while overexpression of the K353Q/L356A α1A 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 SMD4JM in α1A adrenoceptor-mediated signaling.