: 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. ? ?
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