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

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Title: Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis
Author: Hays, Lori Beth
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /407
Date: 2004-05-04

Citation

Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /407 .

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