Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA

We have investigated processing of monkey pro-opiomelanocortin (POMC) following transfection into heterologous neuronal Neuro 2A (N2A) cells. In several separately transfected stable cell lines (termed N2A/POMC2-like; n = 4), POMC was processed to beta E only, by direct cleavage from the precursor. Thus, these cell lines did not produce beta E in the orderly manner observed in the pituitary, that is, via the intermediate peptide beta LPH. Analysis of one representative N2A/POMC2 cell line revealed that the extent of processing to beta E appeared to be negatively correlated with precursor expression level, suggesting that the processing enzyme(s) in these cells was present in limiting amounts. Northern analysis of PC1 and PC2, two recently cloned processing enzymes, showed that N2A/POMC2 cells expressed low levels of PC2 mRNA, but no detectable PC1 mRNA. These data suggest that (1) the order of processing observed in the pituitary is not exclusively determined by tertiary folding of the precursor, but rather by the complement of processing enzymes in a particular cell, and (2) if PC2 is responsible for POMC processing in N2A/POMC2 cells, this enzyme, expressed in limiting amounts, appeared to show selectivity for the beta E amino terminal processing site.