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要求信号复合体的极化组织和功能。了解细胞微域中复合体内信号蛋白的组装和调节是这一提议的中心主题。基于分泌细胞中钙信号蛋白的极化表达，GBy直接调节IP3Rs通道活性的发现，RGS蛋白和GPCRs的第三胞内环(31L)与支架蛋白SPINOPHILIN的结合，Hmer蛋白在PM/ER连接处TRPC通道和IP3R之间的中介通讯中的作用，以及PERK1-/-和Misti-/-小鼠中钙离子转运蛋白和细胞器的错误定位，我们将通过I.门控IP3Rs(及其与胰腺腺泡中钙信号的相关性)来验证中心假说。通过确定参与调控的IP3Rs和G()结构域及其相互作用机制，将对GPCRs介导的这一新的钙信号转导模式进行研究。更重要的是，结构域和多肽及其突变将被用来确定这种钙信号模式对胰腺腺泡细胞钙信号的意义。2.研究SPL在调节RGS蛋白和GPCRs之间的通讯中的作用，这将通过提纯与RGS蛋白相互作用的SPL结构域、SPL在RGS蛋白GAP活性中的作用以及找出干扰SPL与RGS蛋白和GPCRs相互作用的显性负离子来实现。这些工具将被用来检查SPL相互作用在体内的作用。在第二种方法中，M3R 31L中的结构性活跃和信号受损的突变体将被用来进一步定位RGS蛋白和GPCR之间的通讯位点。3.研究Hmer蛋白在PM/ER连接的钙信号转导中的作用。这将通过研究HOMERS在通过IP3Rs和脂类在胰腺腺泡中表达的TRPCI、TRPC3和TRPC6的激活中的作用来实现。在另一种方法中，将在荷马I-/-小鼠的胰腺腺泡中表征钙信号。4.研究PERK1-I-和MIST1-I-小鼠的钙信号转导机制。钙转运蛋白和细胞器在这些小鼠的胰腺腺泡中定位错误，这为我们提供了第一个机会来研究细胞微域中组装的钙信号复合体在极化钙波的启动、传播和产生中的作用。更重要的是，突变小鼠的胰腺发育具有胰腺炎的时间形态特征。因此，这些小鼠可能为研究钙信号在慢性胰腺炎中的作用提供了一个动物模型。我相信，我的实验室中可用的工具、人员的专业知识以及我们建立的合作关系将使我们能够实现我们的目标，并提供与细胞信号转导以及胰腺功能和功能障碍普遍相关的见解。

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.