The Effectiveness of the Prohormone Convertases PC1(3) and PC2 in the Processing of Neurotensin in Transfected PC 12 Cells
Ahn, Susan C.
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The prohormone converting enzymes PC1 and PC2 have been studied in a wide variety of cell in order to characterize their mechanisms in the processing of prohormones. In a novel approach to see the comparative proteolytic effects of the enzymes on an endogenous marker, rat pheochromocytoma PC12 tumor cells were used. The cells were transfected with either PC1(3) or PC2 mouse cDNAs, with normal PC12 cells serving as a control, and subjected to a drug regimen consisting of forskolin, dexamethasone, and nerve growth factor, to cause expression of a manufactured, but normally unprocessed peptide, pro-neurotensin. High-performance gel permeation size-fractionated cellular extracts were subjected to a modified radioimmunoassay. This technique was refined under this study to quantitate both neurotensin and pro-neurotensin using a pepsin digestion to release the free immunoreactive molecule from its precursor. PC12 cells showed a larger amount of cleaved or partially cleaved hormone (-30%) than seen in previous studies with the same drug treatment. PC12/PC2 transfected cells showed only marginal activity in comparison with the controls (-31%). PC1 displayed a significant amount of enzymatic activity in the transfected cells (-61%); however, no cells displayed complete processing of all prohormone produced. Thus PC1 is observed to be more effective in the processing of neurotensin within the adrenal-based PC12 cells, while PC2 is relatively inefficient. This study confirms the suggestion that the expression of a converting enzyme and an appropriate substrate together within any cell may not be enough for processing. Although the tumorous nature of PC12 cells cause them to lose the ability to express PCs, PC1 is native to non-tumorous chromaffin cells of the adrenal medulla, perhaps causing the increased activity of PC1. The high amount of observed processing of proneurotensin in the PCl2 cells suggests that other endogenous endoproteases, not yet characterized, may be implicated in the proteolytic conversion of neurotensin in the secretory vesicles of the PCl2 cell line.