ENDOPROTEOLYSIS AT TETRABASIC AMINO-ACID SITES IN PROCALCITONIN GENE-RELATED PEPTIDE BY PITUITARY CELL-LINES

The specificity of neuroendocrine prohormone convertases for tetrabasi c amino acid sites was investigated. Mutations were introduced into th e tetrabasic cleavage site of the procalcitonin gene-related peptide ( proCGRP) cDNA and these mutated cDNA's were expressed in AtT-20 cells which predominantly express the endoprotease prohormone convertase-1 ( PC1/3), and in GH3 cells which predominantly express prohormone conver tase-2 (PC2). Mutations were introduced into the proCGRP cDNA which co nverted the wild-type ArgArgArgArg site to LysLysArgArg and ArgArgLysL ys, and the proCGRP variants were stably transfected into AtT-20 and G H3 cells. ProCGRP containing each of the LysLysArgArg permutations wer e efficiently cleaved in both AtT-20 and GH3 cells. Cleavage of LysLys ArgArg in exogenous proCGRP, but not in endogenous POMC, suggests that the specificity of cleavage at tetrabasic sites is not defined solely by the endoproteases expressed by the cell or by the amino acid seque nce at the cleavage site, but is also dependent on the structure of th e propeptide. (C) 1997 Elsevier Science Inc.