TISSUE-SPECIFIC AND SUBSTRATE-SPECIFIC ENDOPROTEOLYTIC CLEAVAGE OF MONKEY PROOPIOMELANOCORTIN IN HETEROLOGOUS ENDOCRINE-CELLS - PROCESSING AT LYS-LYS DIBASIC PAIRS

This study compares the processing of pro-opiomelanocortin (POMC) at t wo Lys-Lys cleavage sites, located in the carboxy-terminal domain of t he precursor; one site marking the amino terminus of beta-melanocyte-s timulating hormone (beta-MSH) and the other in the carboxy-terminus of beta-endorphin (betaE), These comparisons were carried out by transfe cting monkey POMC cDNA into two heterologous cell lines: AtT-20, which endogenously expresses mouse POMC, and Rin m5F, which has been previo usly used as a host for transfected POMC. These cells lines are known to process POMC differently at Lys-Arg residues, though less is known about their Lys-Lys cleavage. Our results have demonstrated both tissu e-specific and site-specific factors controlling Lys-Lys cleavage. The AtT-20 line appears not to perform either Lys-Lys cleavage. Rin m5F c ells, on the other hand, fail to process the site at the carboxy termi nus of betaE (betaE28-29) but do process, to a significant extent, the N-terminal site to beta-MSH. That this differential processing is unl ikely to be due to a POMC conformation which would make the betaE site inaccessible was demonstrated by mutating the sites from Lys-Lys to L ys-Arg. With such mutants, Rin m5F cells fully processed at both locat ions. Interestingly, the mutant Lys-Arg sites were not fully processed by AtT-20 cells. These results are discussed in terms of the compleme nt of processing enzymes expressed in each of the cell lines, as well as the role of residues surrounding the diabasic cleavage sites in det ermining the likelihood of proteolysis.