Numerous signal transduction pathways are dependent upon agonist-induced increases in cytoplasmic free Ca2+. The initial phase of this response is often due to the generation of inositol 1,4,5-trisphosphate and a subsequent release of Ca2+ from the endoplasmic reticulum (ER). The depletion of ER Ca2+ stores results in an influx of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry (SOCE). SOCE is implicated in a remarkable array of biological processes, including T cell activation, the secretion of insulin, vasoconstriction, and heart development. One hypothesis that has emerged to explain the transmission of information regarding ER Ca2+ depletion to the plasma membrane proposes a critical role for a novel diffusible messenger molecule, Ca2+ influx factor (ClF). In the last year data that have been gathered by this group of investigators provide strong support for the conclusion that CIF exists, that it is synthesized upon ER Ca2+ store depletion, and that it is responsible for activation of two distinct store-operated plasma membrane Ca2+ channels. Imaging and patch-clamp techniques for CIF's function have provided unique and independently verified data on mammalian CIF. In addition, this group has determined that Saccharomyces cerevisiae that are genetically deficient in an organellar Ca2+ ATPase also make a CIF that to date is indistinguishable from its mammalian counterpart. This inexpensive and plentiful source of material, the purification schemes for CIF that are well underway, and the assays that have been developed place this group in a unique position to purify and structurally characterize this novel signaling molecule. Experiments also will be performed to define the biosynthetic pathway for generating CIF upon ER Ca2+ store depletion, and to define the mechanism by which CIF activates the two plasma membrane Ca2+ + channels that contribute to SOCE. The information that we gain will be applied to a major health problem, diabetes, which mischaracterized by an underlying impairment of SOCE. The hyperglycemia-induced activation of the hexosamine biosynthetic pathway and/or protein kinase C will be investigated as possible regulators of SOCE. The described approach will involve a cross-disciplinary, multi-investigator initiative that applies biologic, biochemical, and structural procedures to this problem. Successful completion of these studies will provide pivotal information on SOCE and may define novel therapeutic targets for diabetes.
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