HORMONE REGULATION OF (CA++)I IN PANCREATIC ACINAR CELLS

腺泡细胞功能的极化特性要求信号复合物的极化组织和功能。理解细胞微域内信号蛋白复合物的组装是本提案的中心主题。基于我们的初步发现：a) RGS蛋白在Ca2+信号传导中的作用；b) IP3R对ICRAC的调控我们将通过1来检验Ca2+信号蛋白组织成复合物的假设。研究RGS蛋白在赋予信号特异性和Ca2+信号复合物组装中的作用。这将通过找到负责受体识别的n端序列来实现。RGS盒和n端信号复合物的定位和分离。研究天然胰腺RGS蛋白在Ca2+振荡中的作用。这些研究将揭示RGS蛋白在Ca2+信号传导中的作用，并通过鉴定分离的复合物中的已知和新蛋白来确定复合物的组成。2. 探索Ca2+信号复合物在PM/ER结-耦合通过ICRAC。为此，我们将确定新发现的微型Ca2+通道Imin是否是ICRAC，并使用删除突变来确定IP3R中调节Imin和ICRAC类似物Htrp3的结构域。该结构域将被用作确定其在Htrp3和Imin中的结合伙伴的诱饵。这些研究将评估耦合假设的有效性，以电容Ca2+进入和PM/ER连接在Ca2+信号复合物组装中的作用。3. SERCA3负/负小鼠和Ca2+信号复合物。组织Ca2+信号复合物的另一种方法是探索SERCA3在Ca2+信号传导中的作用。为了确定SERCA3负/负细胞中受体选择性Ca2+信号传导受损的机制，我们将比较a) Ca2+信号的生化、运输和时空特性b)信号复合体中m3受体的偶联以及c) ER Ca2+在WT和SERCA3负/负细胞中的储存和行为。这些研究将为Ca2+信号复合物的自主功能提供最终的证据。4. 目标骆驼的细胞微域探测。ER-cameleon和融合到VAMP2和Syncollin的N和C末端的cameleon将被包装成腺病毒并感染到腺泡细胞。这些结构应该探测内质网Ca2+和[Ca2+]信号复合物旁边的动力学，以确定它们在[Ca2+]局部控制中的作用。我相信，在之前的拨款期间开发的工具，我的实验室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和外分泌细胞功能一般相关的Ca2+信号的新水平的理解。

The polarized nature of acinar cell function requires polarized organization and function of signaling complexes. Understanding assembly of signaling proteins into complexes within cellular microdomains is the central theme of this proposal. Based on our preliminary findings of: a) the role of RGS proteins in Ca2+ signaling; and b) the regulation of ICRAC by IP3R we will test the hypothesis of organization of Ca2+ signaling proteins into complexes by 1. Study the role of RGS proteins in conferring signaling specificity and assembly of Ca2+ signaling complexes. This will be achieved by finding the N-terminal sequence responsible for receptor recognition. Mapping and isolation of signaling complexes with RGS box and the N-terminus. Study the role of native pancreatic RGS proteins in Ca2+ oscillations. These studies will reveal the role of RGS proteins in Ca2+ signaling and determine complexes composition by identifying known and novel proteins in the isolated complexes. 2. Explore Ca2+ signaling complexes at the PM/ER junction - Coupling through ICRAC. For that we will determine if the newly discovered miniature Ca2+ channel Imin is ICRAC and use deletion mutations to identify the domain in IP3R that regulate Imin and the ICRAC analogue Htrp3. This domain will be used as a bait to identify its binding partner in Htrp3 and maybe to Imin. These studies will assess the validity of the coupling hypothesis to capacitative Ca2+ entry and the role of PM/ER junction in assembly of Ca2+ signaling complexes. 3. SERCA3 minus/minus mice and Ca2+ signaling complexes. An alternative approach to organization of Ca2+ signaling complexes is to probe the role of SERCA3 in Ca2+ signaling. To determine the mechanism of receptor-selective impaired Ca2+ signaling in SERCA3 minus/minus cells we will compare a) the biochemical, transport and spatiotemporal properties of Ca2+ signaling b) the coupling of m3 receptors in signaling complexes and c) storage and behavior of ER Ca2+ in WT and SERCA3 minus/minus cells. These studies will provide the ultimate proof for the autonomous functioning of Ca2+ signaling complexes. 4. Prob cellular microdomains with targeted cameleons. ER-cameleon and cameleons fused to the N and C termini of VAMP2 and Syncollin will be packaged into adenoviruses and infected into acinar cells. These constructs should probe dynamics of ER Ca2+ and the [Ca2+] next to signaling complexes to determine their role in local control of [Ca2+]. I believe that the tools developed in the previous grant period, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide a new level of understanding of Ca2+ signaling of general relevance to cell signaling and to the function of exocrine cells.