Our long term goal is to define the molecular mechanism of store-operated Ca2+ entry (SOCE) in vascular smooth muscle cells (SMC) and other cell types. Recently we identified Ca2+-independent phospholipase A2? (iPLA2?) as a novel determinant of SOCE, which was successfully confirmed by others. We discovered a novel plasma membrane-delimited cascade of reactions involving CIF-induced displacement of inhibitory CaM from iPLA2? which leads to its activation and production of lysophospholipids that in turn activate SOC channels. The importance of iPLA2? was fully confirmed in a recent screen of Drosophila genes, in which iPLA2?, STIM1 and Orai1 (but not TRP) were found to play equally important roles in SOCE, but the mechanisms of their molecular and functional interactions are yet to be determined. Our preliminary studies strongly suggest that iPLA2? may be a key intermediate between STIM1 and Orai1. Based on the careful analysis of the most recent discoveries in the field, critical evaluation of different models, and our extensive preliminary findings, we propose a novel model of SOCE complex (SOCEplex) that brings a new dimension to SOCE mechanism, unifies conformational coupling and diffusible messenger ideas, involves STIM1, CIF, iPLA2? and Orai1, and offers their new structural and functional arrangement. The main hypothesis of this proposal is that iPLA2? is a central multifunctional element of the SOCEplex, and serves as a plasma membrane anchor for endoplasmic reticulum STIM1, acceptor of the signal from depleted stores and transducer of the signal to Orai1. Novel model of SOCE will be tested in primary vascular SMC and model cell lines using our integrative approach, which involves advanced molecular, biochemical, imaging, electrophysiological and functional methods. All approaches and methods have been established and successfully used in the PI's lab. The feasibility of our new hypothesis and success of the proposed studies are fully supported by extensive preliminary data and expertise of the PI and collaborators.
Specific Aims of this proposal are:
Aim 1 : To establish iPLA2? as a molecular partner of STIM1 and Orai1, and a keystone of SOCEplex. We will assess alternative models of SOCE, establish a new role of iPLA22 as a linker between STIM1 and Orai1, identify the molecular mechanism of iPLA2? interaction with STIM1 and its role in SOCE, determine the mechanism of iPLA2? oligomerization and identify molecular domains in iPLA2? that are crucial for signal transduction in SOCEplex.
Aim 2 : To establish the roles of CIF and LysoPL in signal transduction within SOCEplex. We will confirm the role of CIF in signal transduction from depleted stores to plasma membrane, determine the role of lipid rafts, and assess LysoPL-mediated mechanism of Orai1 activation.
Our long term goal is to define the mechanism of store-operated Ca2+ entry (SOCE) pathway that is crucial for a wide variety of physiological functions. The goals of this proposal are to establish the role of specific plasma membrane-bound isoform of Ca2+-independent phospholipase A2 as a crucial component of SOCE machinery, and to demonstrate that it serves as a central molecular, structural and functional element of SOCE complex (SOCEplex) that transduces the signal from depleted stores to plasma membrane channels. The feasibility of our novel hypothesis is fully supported by extensive preliminary data and advanced expertise of PI's lab.
|Thompson, Melissa D; Mei, Yu; Weisbrod, Robert M et al. (2014) Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery. J Biol Chem 289:19907-16|
|Bolotina, Victoria M (2012) Orai1, STIM1, and iPLA2* determine arterial vasoconstriction. Arterioscler Thromb Vasc Biol 32:1066-7|
|Yang, Bo; Gwozdz, Tomasz; Dutko-Gwozdz, Joanna et al. (2012) Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells. Am J Physiol Cell Physiol 302:C748-56|
|Gwozdz, Tomasz; Dutko-Gwozdz, Joanna; Schafer, Claudia et al. (2012) Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators. J Biol Chem 287:22865-72|
|Csutora, Peter; Peter, Krisztina; Kilic, Helena et al. (2008) Novel role for STIM1 as a trigger for calcium influx factor production. J Biol Chem 283:14524-31|
|Gwozdz, Tomasz; Dutko-Gwozdz, Joanna; Zarayskiy, Vladislav et al. (2008) How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation? Am J Physiol Cell Physiol 295:C1133-40|
|Park, Kristen M; Trucillo, Mario; Serban, Nicolas et al. (2008) Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries. Am J Physiol Heart Circ Physiol 294:H1183-7|
|Smani, Tarik; Patel, Tina; Bolotina, Victoria M (2008) Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs. Am J Physiol Cell Physiol 294:C1499-508|
|Zarayskiy, Vladislav; Monje, Francisco; Peter, Krisztina et al. (2007) Store-operated Orai1 and IP3 receptor-operated TRPC1 channel. Channels (Austin) 1:246-52|
|Csutora, Peter; Zarayskiy, Vladislav; Peter, Krisztina et al. (2006) Activation mechanism for CRAC current and store-operated Ca2+ entry: calcium influx factor and Ca2+-independent phospholipase A2beta-mediated pathway. J Biol Chem 281:34926-35|