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 Ca2+ 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 17 beta-estradiol (E-2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca2+ and Ca2+-CaM indicated that E2 also increases free Ca2+-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 a, the plasma membrane Ca2+-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor a and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-G(beta gamma) preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca2+ efflux is reversed by E-2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca2+ signals and promote Ca2+-dependent CaM interactions with other CaM targets. Consequently, E-2 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, E-2 generates a feedforward loop via GPER/GPR30, which enhances Ca2+/CaM signals and functional linkage in the endothelial CaM target network.