The polarized nature of pancreatic acinar cell functions require polarized organization and function of signaling complexes. Understanding assembly and regulation of signaling proteins within complexes in cellular microdomains is the central theme of this proposal. Based on the findings of polarized expression of Ca2+ signaling proteins in secretory cells, the discovery of direct regulation of IP3Rs channel activity by GBy, binding of RGS proteins and the third intracellular loop of GPCRs (31L) to the scaffolding protein spinophilin, the role of Homer proteins in mediated communication between TRPC channels and IP3Rs at the PM/ER junction and the mislocalization of Ca2+ transporting proteins and organelles in PERK1-/- and MISTI -/- mice, we will test the central hypothesis by I. Study gating of IP3Rs by G(( and its relevance to Ca 2+ signal in pancreatic acini. This new mode of Ca 2+ signaling by GPCRs will be examined by identifying the IP3Rs and G(( domains that mediate the regulation and the mechanism of their interaction. More important, the domains and peptides and mutations thereof will be used to determine the significance of this mode of Ca2+ signaling to Ca2+ signaling by pancreatic acini. 2. Study the role of spinophilin (SPL) in mediating communication between RGS proteins and GPCRs, This will be achieved by refining the SPL domains that interact with RGS proteins, the role of SPL in RGS proteins GAP activity and identify dominant negatives that interfere with the interaction of SPL with RGS proteins and GPCRs. These tools will be used to examine the role of SPL interactions in vivo. In a second approach, constitutively active and signaling impaired mutants in the 31L of the M3R will be used to further map the site of communication between RGS proteins and GPCR. 3. Study the role of Homer proteins in Ca 2+ signaling at the PM/ER junction. This will be achieved by examining the role of Homers in activation of TRPCI, TRPC3 and TRPC6 that are expressed in pancreatic acini by IP3Rs and by lipids. In another approach, Ca2+ signaling will be characterized in pancreatic acini of Homer I -/- mice. 4. Characterize Ca 2+ signaling in PERK1 -I-and MIST1-I- mice. Ca2+ transporting proteins and organelles are mislocalized in pancreatic acini of these mice, offering us the first opportunity to examine the role of assembly of Ca2+ signaling complexes in cellular microdomains in the initiation, propagation and generation of polarized Ca2+ waves. More important, the pancreas of the mutant mice develops with time morphological characteristics of pancreatitis. Hence, these mice may provide an animal model to study the role of aberrant Ca 2+ signaling in chronic pancreatitis. I believe that the tools available in my lab, the expertise of the personnel and the collaborations we established will allow us to achieve our goals and provide insights of general relevance to cell signaling and to the function and dysfunction of the pancreas.
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