Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis




Hays, Lori Beth

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Insulin exocytosis is a complex, regulated process involving numerous exocytotic proteins to coordinate the release of insulin. Syncollin has been implicated in zymogen granule exocytosis in acinar cells. It was hypothesized that either syncollin or a ‘syncollin-like’ protein may be expressed in β-cells and influence insulin exocytosis. Adenoviral mediated expression of either long or short forms of syncollin in isolated islets and INS-1 cells showed both forms underwent N-terminal signal peptide cleavage to yield the same 14kD mature protein. Immunofluorescence revealed that adenovirally-expressed syncollin was specifically targeted to the ß-granule lumen. In perifused islets, syncollin expression significantly inhibited first-phase glucose-induced insulin secretion compared to AdV-GFP infected islets. GLP-1 and glyburide potentiation of insulin secretion was inhibited; whereas constitutive secretion and insulin content were normal in syncollin-infected islets indicating syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production or secondary stimulus-coupling signals. Thus, syncollin likely inhibited the distal stages of insulin exocytosis providing the first evidence that an intragranular protein is capable of influencing regulated insulin secretion. Syncollin fluorescent fusion proteins were localized to ß-granules, but did not influence insulin secretion implicating these chimeras as ß-granule specific markers for emerging imaging technology. Real-time confocal microscopy demonstrated syncollin-GFP could be used to examine spatiotemporal dynamics of exocytosis. Furthermore, consecutive infection of syncollin-GFP and syncollin-dsRFP labeled distinct pools of β-granules. Expression of syncollin was not identified in β-cells; however, a 10Kd ‘syncollin-like’ protein was expressed, which when sequenced corresponded to FXYD6. Comparison of syncollin and FXYD6 protein structure revealed several conserved domains, indicating syncollin is likely a pseudo-FXYD family member. FXYD6 was the only FXYD protein endogenously expressed in β-cells, which localized to distinct regions of the plasma membrane. Overexpression of FXYD6-Myc enhanced β-granule transport to distinct regions of the plasma membrane that also expressed FXYD6; however, there was no significant effect on glucose-stimulated insulin secretion in isolated islets. SiRNA-mediated reduction of FXYD6 resulted in no obvious changes in β-granule distribution; however, β-granule movement during glucose stimulation was erratic and misdirected. These data implicate FXYD6 as a molecular beacon on the plasma membrane guiding β-granules to the active site of exocytosis.

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