Acinar Cell Biology and Pancreatic Disease

描述（由申请人提供）：在正常和病理生理条件下，Ca2+在调节胰腺腺泡细胞功能中的关键作用是公认的，然而，响应Ca2+升高而发生的分子变化在很大程度上是未知的。本研究提出了一种新的机制，即胞质Ca2+在腺泡细胞的分泌和内吞途径中调节磷脂和相关调节蛋白的运输。钙反应性热稳定蛋白（CRHSP-28）是腺泡细胞分泌途径的关键调控分子。CRHSP-28受细胞Ca2+变化的高度调节，其Ca2+依赖性表明：1)调节消化酶分泌；2)与囊泡运输蛋白膜联蛋白VI的相互作用；3)丝氨酸磷酸化通过Ca2+/钙调素依赖性蛋白激酶II，触发从膜相关复合体释放CRHSP-28。本提案的主要目的是验证CRHSP-28在腺泡中作为Ca2+传感器促进和稳定膜运输和消化酶分泌所必需的关键蛋白相互作用的假设。在Specific Aim 1中，将进行实验来解决CRHSP-28与膜联蛋白VI相互作用的概念，以指导CRHSP-28与支持腺泡细胞分泌功能所必需的顶细胞质中新形成的内体的结合。靶向CRHSP-28上膜联蛋白VI结合域的位点特异性突变体将在腺泡中表达，并确定其对酶原分泌和膜运输的影响。Specific Aim 2将利用改变主要CaM激酶II磷酸化位点、丝氨酸100和磷酸化特异性抗体的CRHSP-28突变体来测试磷酸化通过将其从膜结合状态取代来抑制CRHSP-28功能的理论。在Specific Aim 3中，实验将提出CRHSP-28存在于不同于酶原颗粒的根尖膜运输途径中，并将重要的调节分子运送到腺泡分泌所必需的根尖膜的假设。阐明CRHSP-28调节外分泌功能的分子机制，应该为了解腺泡细胞分泌途径的Ca2+依赖性提供有价值的见解，这对于制定针对外分泌胰腺疾病的治疗策略至关重要。

DESCRIPTION (provided by the applicant): The pivotal role of Ca2+ in regulating pancreatic acinar cell function under normal and pathophysiological conditions is well established, however, the molecular changes that occur in response to elevated Ca2+ are largely unknown. This proposal addresses a novel mechanism by which cytosolic Ca2+ modulates the trafficking of phospholipids and associated regulatory proteins in the secretory and endocytic pathways in acinar cells. Calcium responsive heat-stable protein (CRHSP-28) is a key regulatory molecule in the secretory pathway of acinar cells. CRHSP-28 is highly modulated by changes in cellular Ca2+ as indicated by its Ca2+-dependent: 1) regulation of digestive enzyme secretion; 2) interaction with the vesicle trafficking protein annexin VI; and 3) serine phosphorylation via Ca2+/calmodulin-dependent protein kinase II, which triggers the release of CRHSP-28 from a membrane associated complex. The primary objective of this proposal is to test the hypothesis that CRHSP-28 acts as Ca2+-sensor in acini to promote and stabilize key protein interactions that are necessary for membrane trafficking and digestive enzyme secretion. In Specific Aim 1 experiments will be conducted to address the concept that the interaction of CRHSP-28 with annexin VI functions to direct CRHSP- 28 association to newly formed endosomes in the apical cytoplasm that are necessary to support acinar cell secretory function. Site specific mutants targeting the annexin VI binding domain on CRHSP-28 will be expressed in acini and their effects on zymogen secretion and membrane trafficking determined. Specific Aim 2 will utilize CRHSP-28 mutants that alter the major CaM kinase II phosphorylation site, serine 100, and phospho-specific antibodies to test the theory that phosphorylation acts to inhibit CRHSP-28 function by displacing it from a membrane-bound state. In Specific Aim 3 experiments will address the hypothesis that CRHSP-28 is present in an apical membrane trafficking pathway that is distinct from zymogen granules and acts to shuttle important regulatory molecules to the apical membrane that are necessary for acinar secretion. Elucidation of the molecular mechanism by which CRHSP-28 modulates exocrine function should provide valuable insight into the Ca2+-dependent nature of the secretory pathway in acinar cells, which is essential for the development of therapeutic strategies aimed at the treatment of exocrine pancreatic disease.