Endothelial regulation of calmodulin expression and eNOS-calmodulin interaction in vascular smooth muscle.

Calmodulin (CaM) is a Ca2+ sensor protein that is required for numerous vascular smooth muscle cell (VSMC) functions. Since CaM is not expressed enough for its many target proteins, factors that modulate its expression and interactions with targets in VSMCs can have extensive effects on vascular functions. VSMCs receive many regulatory inputs from endothelial cells (ECs). However, it is unknown if ECs regulate vascular functions via controlling expression of CaM and its interactions in VSMCs. In this work, we tested the hypothesis that ECs also affect VSMC signaling via regulation of CaM expression and interactions with its target proteins in VSMCs. Using ECs and VSMCs isolated from the same vessels and grown in a co-culture system, we observed that the presence of proliferating ECs significantly upregulates total CaM expression in VSMCs. An imaging module was devised to concurrently measure free Ca2+ and CaM levels in VSMCs in co-culture with ECs. Using indo-1/AM and a CaM biosensor built from a modified CaM-binding sequence of endothelial nitric oxide synthase (eNOS), this system revealed that in response to a generic Ca2+ signal, free Ca2+-bound CaM level is enhanced ~ threefold in VSMCs in co-culture with proliferating ECs. Interestingly, VSMCs express eNOS and eNOS-CaM association in response to the same Ca2+ stimulus is also enhanced ~ threefold in VSMCs co-cultured with ECs. Mechanistically, the endothelium-dependent upregulation of CaM in VSMCs is not affected by inhibition of NO production or endothelin receptors but is prevented by inhibition of vascular endothelial growth factor receptors. Consistently, VEGF-A level is upregulated in VSMCs co-cultured with proliferating ECs. These data indicate a new role of the endothelium in regulating vascular functions via upregulating CaM and its interactions in VSMCs.

Novel regulations of the angiotensin II receptor type 1 by calmodulin.

The angiotensin II receptor type 1 (AT1R) mediates many Ca2+-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca2+ signals in cells. Two locations on the receptor's submembrane domains (SMD) 3 and 4 are known to interact with CaM. However, the binding sites for CaM, biochemical properties of the interactions, and their functional impact are not fully understood. Using a FRET-based screening method, we identified a new binding site for CaM on SMD2 (a.a. 125-141), in addition to SMD3 and the juxtamembranous region of SMD4 (SMD4JM, a.a., 309-327). Simultaneous measurements of CaM binding and free Ca2+ show that the interactions are Ca2+-dependent, with disparate Kd and EC50(Ca2+) values within the physiological range of cytoplasmic Ca2+. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215-242) and has an EC50(Ca2+) value in the range of resting cytoplasmic Ca2+, suggesting AT1R-CaM interaction can occur in resting conditions in cells. AngII induces robust ERK1/2 phosphorylation in primary vascular smooth muscle cells. This effect is suppressed by AT1R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT1R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca2+ signals in cells expressing wild-type AT1R. These signals are reduced in cells expressing mutant AT1R with reduced CaM binding at SMD3 or SMD4JM, but are very slow-rising, low amplitude signal in cells expressing AT1R with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT1R can occur at various domains, with different affinities, at different physiological Ca2+ levels, and are important for AT1R-mediated signaling.

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

The G protein-coupled estrogen receptor 1 (GPER, formerly also known as GPR30) modulates many Ca2+-dependent activities in endothelial cells. However, the underlying mechanisms are poorly understood. We recently reported that GPER acts to prolong cytoplasmic Ca2+ signals by interacting with and promoting inhibitory phosphorylation of the plasma membrane Ca2+-ATPase. In the present study, we examined the role of GPER activation in modulating store-operated Ca2+ 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 Ca2+ signals in primary endothelial cells. G-1 dose-dependently inhibits thapsigargin-induced SOCE measured by the Mn2+ 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 Ca2+ efflux, the current data contribute to a model in which GPER acts to clamp agonist-induced cytoplasmic Ca2+ 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 Ca2+ signals.

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.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17ß-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities.

Biosensor-based approach identifies four distinct calmodulin-binding domains in the G protein-coupled estrogen receptor 1.

The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+)-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s), we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs). Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+)-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+) indicators identified separate Ca(2+) sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+) response, representing two distinct species (SMD1 sp1 and SMD1 sp2) with drastically different Ca(2+) sensitivities. The Ca(2+) sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+)) values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER-dependent signaling at the receptor level through multiple interaction sites. FRET biosensors represent a simple method to identify unknown calmodulin-binding domains in G protein-coupled receptors and to quantitatively assess binding properties.