Enhanced Linkage in the Vascular Calmodulin Network by Estrogen via a Feedforward at the G Protein‐Coupled Estrogen Receptor 1 (GPER/GPR30)

Plasma estrogen is strongly linked to cardiovascular health. Calmodulin (CaM) is required for the activities of numerous proteins but is insufficiently expressed for all its targets, leading to competition among CaM targets. It is completely unknown if estrogen modulates cardiovascular functions via the CaM network. We observed in endothelial cells that chronic estrogen treatment (CE 2 T) increases total and free CaM. Chronic treatment with G‐1, an agonist of the estrogen‐sensitive receptor GPER/GPR30 mimics this effect, while agonists of ERa or ERb do not. ICI182780, an ERa/ERb antagonist and GPER/GPR30 agonist, also increases CaM level. CE 2 T increases CaM binding to different categories of CaM targets, including the plasma membrane Ca 2+ ‐ATPase (PMCA), eNOS, ERa, and GPER/GPR30 itself. For GPER/GPR30, CaM antagonism or CaM binding‐negating mutations in the receptor's multiple CaM‐binding domains prevent GPER/GPR30‐mediated ERK1/2 phosphorylation. For PMCA, CE 2 T‐induced stimulation of activity through enhanced CaM binding is masked by Src‐dependent phosphorylation. These effects sustain cytoplasmic Ca 2+ for enhanced interactions between CaM and other targets. For eNOS, CE 2 T doubles CaM binding. Kinetic modeling using in‐cell and in vitro data allowed comparison of CE 2 T's promotion of eNOS point activity and NO accumulation via effects on determinants of eNOS function, including Ca 2+ , CaM, and phosphorylation. Our data indicate that CE 2 T improves endothelial functions via a feed‐forward mechanism in which CaM is upregulated through GPER/GPR30 activation, leading to enhanced CaM binding and functional linkage in the network of CaM‐binding proteins.