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.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17ß-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gßγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.