Suppression of store-operated Ca2+ entry by activation of GPER: contribution to a clamping effect on endothelial Ca2+ signaling

Abstract

The G protein-coupled estrogen receptor 1 (GPER, formerly also known as GPR30) modulates many Ca-dependent activities in endothelial cells. However, the underlying mechanisms are poorly understood. We recently reported that GPER acts to prolong cytoplasmic Ca signals by interacting with and promoting inhibitory phosphorylation of the plasma membrane Ca-ATPase. In the present study, we examined the role of GPER activation in modulating store-operated Ca entry (SOCE) via effects on the stromal interaction molecule 1 (STIM1). GPER activation by agonist G-1 reduces the peak but prolongs the plateau of bradykinin-induced Ca signals in primary endothelial cells. G-1 dose-dependently inhibits thapsigargin-induced SOCE measured by the Mn quenching method. GPER heterologous expression reduces SOCE, which is further pronounced by G-1 treatment. Consistently, GPER gene silencing in endothelial cells is associated with an increase in SOCE. Treatment with G-1 reduces puncta formation by STIM1 triggered by the activation of SOCE. The effect of GPER activation to inhibit SOCE is not affected by combined nonphosphorylatable substitutions at serines 486 and 668 on STIM1, but is substantially reduced by similar substitutions at serines 575, 608 and 621. Taken together with our recently reported inhibitory actions of GPER on Ca efflux, the current data contribute to a model in which GPER acts to clamp agonist-induced cytoplasmic Ca signals. Kinetic modeling based on current and reported data is used to estimate the overall effect of GPER activation on point activity of endothelial nitric oxide synthase during the time course of agonist-induced total Ca signals.

Reciprocality Between Estrogen Biology and Calcium Signaling in the Cardiovascular System

“…These effects may be attributed to inhibition of both VDCE and SOCE, as α 1 adrenoceptor agonists can activate both ( 77 ). GPER-mediated inhibition of SOCE has been shown in ECs, where G-1 (10 −8 -10 −6 M) suppresses SOCE induced by thapsigargin or bradykinin ( 203 ). Interestingly, the observations that in the absence of any treatment with agonists, thapsigargin-induced SOCE is increased by 80% in GPER-knockdown ECs and is reduced by 40% in GPER-overexpressing HEK293 cells implicate E 2 -independent mechanisms ( 203 ).…”

“…How E 2 /GPER suppresses SOCE seems to involve STIM1. G-1 treatment prevents thapsigargin-induced STIM1 puncta, indicating inhibition of STIM1's association with the Ca 2+ channel; and Ser575/608/621Ala mutations of STIM1 reduce the inhibitory effect of G-1 ( 203 ). Consistently, E 2 inhibits Ser575 STIM1 phosphorylation in bronchial epithelial cells, thus suppressing STIM1 mobility and SOCE ( 204 ).…”

“…E 2 -independent mechanisms are indicated by the findings that (1) GPER constitutively interacts with PMCA4b via the anchoring action of PSD-95 at their C-terminal PDZ-binding motifs; (2) overexpression of GPER decreases PMCA activity; (3) GPER knockdown promotes PMCA activity; and (4) PSD-95 knockdown or truncation of the PDZ-binding motif on GPER releases GPER–PMCA association and promotes PMCA activity ( 135 ). Functionally , these mechanisms collectively prolong agonist-induced Ca 2+ signal and enhance eNOS activity in ECs ( 135 , 170 , 203 ). Consistent with suppressed Ca 2+ efflux, the Ca 2+ signals stimulated by E 2 and the GPER agonist G-1 in cells overexpressing GPER reported by various laboratories display much more prolonged plateau phases compared to Ca 2+ signals in cells not overexpressing GPER or those stimulated by other agonists such as ATP or bradykinin ( 160 , 162 , 164 , 175 ).…”

“…Notably, its bi-phosphorylation at Ser617 and Ser1179 promotes NO production by increasing the Ca 2+ sensitivity for both CaM binding and enzyme activation, reducing their respective EC 50 (Ca 2+ ) values to ~0.7 × 10 −7 and 1.3 × 10 −7 M, thus rendering the synthase active at resting cytoplasmic Ca 2+ ( 189 ). E 2 and GPER (1) prolong endothelial cytoplasmic Ca 2+ signal by inhibiting Ca 2+ efflux ( 135 , 170 ), (2) promote eNOS phosphorylation at Ser617 and Ser1179 ( 170 , 198 ), (3) increase CaM expression and eNOS–CaM interaction ( 170 ), and (4) suppress endothelial SOCE ( 203 ). When we incorporate these effects into a verified sequential “CaM binding eNOS activation” model ( 189 , 190 ), eNOS activity and NO accumulation are shown to substantially increase across the time course of bradykinin-induced Ca 2+ signal in ECs by treatment with G-1 ( 203 ).…”

“…E 2 and GPER (1) prolong endothelial cytoplasmic Ca 2+ signal by inhibiting Ca 2+ efflux ( 135 , 170 ), (2) promote eNOS phosphorylation at Ser617 and Ser1179 ( 170 , 198 ), (3) increase CaM expression and eNOS–CaM interaction ( 170 ), and (4) suppress endothelial SOCE ( 203 ). When we incorporate these effects into a verified sequential “CaM binding eNOS activation” model ( 189 , 190 ), eNOS activity and NO accumulation are shown to substantially increase across the time course of bradykinin-induced Ca 2+ signal in ECs by treatment with G-1 ( 203 ). Importantly, major contributions to this outcome include the increases in CaM binding, phosphorylation, Ca 2+ sensitivity, and duration of Ca 2+ signals due to Ca 2+ efflux inhibition, but little or no effect of the inhibition of SOCE ( 203 ), due obviously to the synthase's high Ca 2+ sensitivity ( Figure 3 ).…”