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 net-
Cardiovascular functions are mediated by multiple 7-pass transmembrane receptors whose activation promotes contraction or relaxation of the tissues. The α 1 adrenoceptor type 1A plays important roles in the control of vascular tone and myocardial contractility via Ca 2+ -dependent actions. Here, using novel FRET-based biosensors, we identified a novel Ca 2+ -dependent interaction between calmodulin (CaM) and the human α 1A adrenoceptor at the juxtamembranous region of its 4 th submembrane domain (SMD4 JM , a.a. 333-361). SMD4 JM houses the known nuclear localization signal of α 1A adrenoceptor (NLS, a.a. 334-349). We found that NLS itself also interacts with CaM, but with lower affinity and Ca 2+ sensitivity, indicating that full interaction between CaM and α 1A receptor in this region requires segment a.a. 333-361. Combined K353Q/ L356A substitutions in the non-NLS segment of SMD4 JM cause a 3.5-fold reduction in the affinity of CaM-SMD4 JM interaction. Overexpression of wild-type α 1A adrenoceptor in cells enhances phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) stimulated by A61603, while overexpression of the K353Q/L356A α 1A receptor mutant significantly reduces this signal. Norepinephrine stimulates intracellular Ca 2+ signals that are higher in cells overexpressing wildtype receptor but lower in cells overexpressing the K353Q/L356A receptor compared to nontransfected cells in the same microscopic environments. These data support a novel and important role for Ca 2+ -dependent CaM interaction at SMD4 JM in α 1A adrenoceptor-mediated signaling.
Reduced estrogen concentrations following menopause are associated with increases in the incidence of cardiovascular disease. Calmodulin (CaM) is required for the activities of numerous cardiac proteins yet is not sufficiently expressed for its binding targets. We have begun to examine the effects of 17b‐estradiol (E2) on the cardiac network of CaM‐binding proteins. Female rats received sham surgery or ovariectomy, followed by treatment with vehicle or E2 for 2 weeks. E2 treatment in sham animals increases the interaction between endogenous CaM and the cardiac α adrenergic receptor type 1A. Surprisingly, ovariectomy substantially increases this interaction, while E2 replacement now reduces it. To examine the effects of these treatments on the population of cardiac CaM‐binding sites that are unsaturated by endogenous CaM, lysate from left ventricle was subjected to saturating Ca2+ concentration and processed through a CaM sepharose column. Flow through contained CaM‐binding sites saturated by endogenous CaM, while sepharose‐bound fraction represented endogenously unsaturated CaM‐binding sites. The fractions eluted from the CaM sepharose were subsequently used in competitive binding assays using purified CaM and a CaM biosensor. E2 treatment in sham animals increases the number of unsaturated CaM‐binding sites. Ovariectomy further increases this number, while E2 treatment now reduces it. The data indicate that E2 treatment exerts opposing effects in the presence and absence of ovaries on specific endogenous CaM‐target associations and the number of endogenously unsaturable Ca2+‐dependent CaM‐binding sites in the heart. These results suggest that estrogen replacement may have unpredictable functional outcomes.Support or Funding InformationSupported by NIH Grant HL112184 to Quang‐Kim Tran and Grants from the Iowa Osteopathic and Educational Research Funds to Sarah Clayton and Quang‐Kim Tran.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The stromal interaction molecule 1 (STIM1) is essential for activation of store‐operated Ca2+entry (SOCE). STIM1 contains in its intra‐endoplasmic reticular (ER) domain a canonical EF hand (cEF) that houses a Ca2+‐binding loop. This loop serves as a Ca2+sensor; Ca2+leaving its binding loop upon ER Ca2+depletion initiates conformational changes leading to STIM1 oligomerization and SOCE activation. We recently reported that activation of the G protein‐coupled estrogen receptor (GPER) clamps intracellular Ca2+signals via multifaceted inhibitory actions on both Ca2+efflux and SOCE; the latter results partly from alteration in phosphorylation at serines 575, 608 and 621 of STIM1. Here, we identified a dynamic interaction between GPER and STIM1 that directly controls the Ca2+‐sensing capacity of STIM1. GPER coimmunoprecipitates with STIM1 in resting primary endothelial cells, an association enhanced by treatment with either GPER agonist G‐1 or activation of SOCE using thapsigargin. This effect is not altered by combined S575/608/621A substitutions. Coimmunoprecipitation analysis using truncated versions of STIM1 showed that GPER associates with the cEF domain. Confocal microscopy identified a GPER's submembrane domain that localizes GPER to the ER. To assess the role of GPER‐STIM1 interaction in controlling STIM1's Ca2+sensing capacity, we generated a series of biosensors that incorporated STIM1's Ca2+‐binding loop in singlet, doublet or triplet. These biosensors faithfully report Ca2+binding to the loop, with tripled dynamic range by the triple‐loop biosensor and loss of Ca2+binding by charge substitutions targeting strategic elements for Ca2+binding. Using peptides corresponding to submembrane domains of GPER and these STIM1 biosensors, we identified a robust Ca2+‐independent interaction between a submembrane domain and the Ca2+‐binding loop. The charge substitutions that abolish Ca2+binding only reduce but do not negate the interaction, indicating that Ca2+and the GPER domain bind to different regions of the loop. Consistently, we have observed that GPER overexpression decreases SOCE and GPER knockdown substantially increases SOCE. These data reveal a new interaction between GPER and STIM1 that directly controls STIM1's Ca2+‐sensing capacity and SOCE activation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In addition to its role in vasoconstriction, the α1A‐adrenergic receptor (α1A‐AR) is increasingly recognized as a rescue mechanism under circumstances where b‐ARs are insufficient to provide stimulus in the myocardium, such as in heart failure. Mechanisms controlling α1A‐AR function therefore have profound impact on cardiovascular functions. Calmodulin (CaM) is the ubiquitous transducer of Ca2+ signals, and has recently been demonstrated to interact with a number of G protein‐coupled receptors. We have observed that in fresh ventricular tissues, α1A‐AR forms a complex with calmodulin in resting condition as well as under α1A‐AR agonism. To identify and characterize the precise interaction domains between CaM and α1A‐AR, we developed novel biosensors that span multiple fragments of the four sub‐membrane domains (SMDs) of α1A‐AR. Responses of these biosensors to Ca2+‐saturated CaM reveals that α1A‐AR directly interacts with CaM at a number of locations with disparate affinities and Ca2+ sensitivities. These data implicate CaM as a novel partner for α1A‐AR and provide background for on‐going studies on the functional impact of these interactions in the control of cardiovascular functions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.