Hormone Regulation of [Ca2+]i in Pancreatic Acinar Cells

描述（由申请人提供）：胰腺腺泡细胞功能的极化性质需要信号复合物的极化组织和功能。了解组装和调节信号蛋白内的复合物在细胞微域是这个建议的中心主题。基于Ca 2+信号蛋白在分泌细胞中的极化表达的发现，通过GBy直接调节IP 3Rs通道活性的发现，RGS蛋白和GPCR的第三环（31 L）与支架蛋白spinophilin的结合，Homer蛋白在TRPC通道和PM/ER连接处的IP 3Rs之间介导的通讯中的作用以及Ca 2+的错误定位在PERK 1-/-和MISTI -/-小鼠中转运蛋白和细胞器，我们将通过I.胰腺腺泡G（（）对IP 3受体的门控及其与Ca 2+信号的关系通过鉴定介导调控的IP 3R和G（）结构域及其相互作用机制，将研究GPCR介导的这种新的Ca 2+信号传导模式。更重要的是，结构域和肽及其突变将用于确定这种Ca 2+信号传导模式对胰腺腺泡的Ca 2+信号传导的意义。2.研究亲棘素（spinophilin，SPL）在介导RGS蛋白和GPCR之间的通讯中的作用，这将通过细化SPL与RGS蛋白相互作用的结构域、SPL在RGS蛋白GAP活性中的作用以及鉴定干扰SPL与RGS蛋白和GPCR相互作用的显性负性来实现。这些工具将用于检查SPL相互作用在体内的作用。在第二种方法中，M3 R的31 L中的组成型活性和信号传导受损的突变体将用于进一步绘制RGS蛋白和GPCR之间的通信位点。3.研究Homer蛋白在PM/ER连接处Ca 2+信号转导中的作用。这将通过检查Homers在TRPCI、TRPC 3和TRPC 6的激活中的作用来实现，TRPC 3和TRPC 6通过IP 3R和脂质在胰腺腺泡中表达。在另一种方法中，将在Homer I -/-小鼠的胰腺腺泡中表征Ca 2+信号传导。4.表征PERK 1-I-和MIST 1-I-小鼠中的Ca 2+信号传导。Ca 2+转运蛋白和细胞器在这些小鼠的胰腺腺泡中被错误定位，为我们提供了第一次机会来研究细胞微区中Ca 2+信号复合物的组装在极化Ca 2+波的启动、传播和产生中的作用。更重要的是，突变小鼠的胰腺具有胰腺炎的时间形态学特征。因此，这些小鼠可能提供了一种动物模型，以研究异常Ca 2+信号在慢性胰腺炎中的作用。我相信，我实验室中可用的工具，人员的专业知识和我们建立的合作将使我们能够实现我们的目标，并提供与细胞信号传导以及胰腺功能和功能障碍相关的见解。

DESCRIPTION (provided by applicant): 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.