Abstract
(Englisch)
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In neuroendocrine cells, hormones are sorted to large dense-core secretory granules. The programme project seeks to understand how prohormones are recognized in the TGN, how immature secretory granules are formed (bud) from the TGN and how such granules are refined to form the mature granule.
The specific aims of the Swiss component are to identify structural domains (or sequences) in proinsulin which allow it to be sorted in the TGN and delivered to nascent (immature) granules, and to follow the fate of proinsulin, insulin and C-peptide in the maturing granule.
Results from Year 04 of funding:
The role of clathrin in the sorting of proinsulin to secretory granules, the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic ß-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain, and green fluorescent protein (EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. To study proinsulin sorting and conversion, and regulated (pro)insulin secretion, cells were pulse-labelled with [3H]leucine (10 minutes) and then subject to 150 minutes chase under basal conditions. Thereafter the cells were incubated for a further 60 minutes chase with a cocktail of secretagogues to stimulate secretion. Chase media and cell extracts were analyzed by reverse-phase hplc to quantify proinsulin, insulin and C-peptide. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway. There was, furthermore, no effect of Hub on proinsulin conversion to insulin (76.2 ± 7.9 vs. 80.7 ± 3.5% conversion in cell extracts after 210 minutes chase for Hub vs. control) or on the percentage of total labelled proinsulin/insulin released in response to the secretagogues (58.5 ± 3.4 vs. 60.4 ± 4.3, Hub vs. control). There was, however, a significant increase in the percentage of C-peptide truncated to des-(27-32)-C-peptide and released during the 150 minutes (basal) chase in cells expressing Hub (10.3 ± 1.25 vs. 5.0 ± 0.2%, Hub vs. control) and more extensive degradation of C-peptide in the stimulated medium (ratio of insulin to intact plus truncated C-peptide 1.28 ± 0.04 vs. 1.10 ± 0.04 for Hub vs. control). The amount of C-peptide released during this same period via the so-called post-granular constitutive route was not affected by expression of Hub. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. It may, however, play a role in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.
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