Endocrine and neuroendocrine cells store peptide hormone and neuropeptides in secretory granules in the regulated secretory pathway while constitutive secretory proteins are directly secreted without intracellular storage. Sorting of the peptide precursors to the regulated secretory pathway is critical for the stimulated release of bioactive peptides but the sorting mechanisms remain largely unknown. Calcium-and low pH-induced aggregation and membrane binding have been proposed to play a role in the sorting of different regulated secretory proteins. However, their relative contributions to sorting is not clear. The long term goal of this research is to determine the roles of membrane binding and protein aggregation for sorting of regulated secretory proteins. Chromagranin A (CgA) is co-stored with peptide hormones in secretory granules of most endocrine and neuroendocrine cells. We have recently identified distinct domains in CgA that may function in membrane binding and aggregation, respectively. Thus, CgA offer a unique opportunity to determine the relative contributions of membrane binding and aggregation to sorting of an individual protein. Based on these observation we propose that separate membrane binding and aggregation sites act as redundant sorting domains for sorting of CgA to the regulated secretory pathways of endocrine cells. To test this hypothesis, the following specific aims are proposed: 1. Determine if the N-terminal domain of CgA plays a role in membrane binding and sorting to the regulated secretory pathway; 2. Determine which part of the C-terminal domain of CgA plays a role in aggregation and sorting to the regulated secretory pathway in neuroendocrine cells. To address these questions, we will delete the putative membrane-binding and aggregation domains and determine the effect on sorting, membrane-binding and aggregation of CgA. Transfer chimeras will be constructed to test if these domains are sufficient for sorting, membrane binding and aggregation in endocrine cells. The proposed studies will provide a framework for understanding the physiology and pathology of how endocrine cells deliver bioactive peptides to the circulation.
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