Abstract
The mammalian subtilisin-like endoproteases furin and PC2 catalyze similar reactions but in different parts of the cell: furin in the trans-Golgi network and PC2 in dense-core granules. To map targeting domains within PC2, chimeras were constructed of the pro-, catalytic, and middle domains of furin with the carboxyl-terminal domain of PC2 (F-S-P) or of the pro- and catalytic domains of furin with the middle and carboxyl-terminal domains of PC2 (F-N-P). Their behavior in stable transfected AtT-20 cells was compared to a furin mutant truncated after the middle domain (F-S), wild-type furin, and with wild-type PC2. F-S-P, F-N-P, and F-S were catalytically active and underwent post-translational proteolysis and N-glycosylation with similar kinetics to wild-type furin. The truncated furin mutant was not stored intracellularly, whereas both chimeras, like PC2, showed intracellular retention and regulated release. Immunofluorescence and immuno-electron microscopy showed the presence of the chimeras and PC2 in dense-cored secretory granules together with proopiomelanocortin immunoreactivity. PC2 was sorted more efficiently than F-S-P, and the inclusion of the middle domain (F-N-P) further enhanced intracellular retention. It is concluded that sorting of PC2 into the regulated pathway depends on its carboxyl terminus. The middle domain may provide additional sorting determinants or a conformational framework for expression of the sorting signal.
Highlights
The mammalian subtilisin-like endoproteases furin and PC2 catalyze similar reactions but in different parts of the cell: furin in the trans-Golgi network and PC2 in dense-core granules
The seven family members currently documented all exhibit a common domain structure characterized by a short unique amino-terminal signal peptide, a moderately conserved pro-domain usually of 70 –90 aa terminating in an endoproteolytic cleavage site comprised of a cluster of basic residues, a highly conserved subtilisin-like catalytic domain of 320 –340 aa, and a well conserved “middle” domain of 110 –130 aa followed by unique carboxyl-terminal domains varying from 50 to several hundred residues in length
The 50-aa carboxyl terminus of PC2 terminates in a putative amphiphatic ␣-helix and is preceded by a stretch of charged aa, a feature which is apparent in PC1 (Smeekens et al 1991) and the unrelated secretory granule metalloexopeptidase carboxypeptidase E (H) (Parkinson, 1990; Mitra et al 1994)
Summary
The mammalian subtilisin-like endoproteases furin and PC2 catalyze similar reactions but in different parts of the cell: furin in the trans-Golgi network and PC2 in dense-core granules. A major limitation of this approach, the case for gene transfer experiments performed with chimeras of heterologous proteins, is that any perturbation of the three-dimensional structure of the protein is likely to mask the activity of a putative sorting motif. To minimize these effects, we have chosen to perform experiments with the family of mammalian subtilisin-like proprotein processing enzymes (reviewed by Hutton, 1990; Steiner et al, 1992; Van de Ven et al, 1993; Halban and Irminger, 1994; Seidah et al, 1994), which includes proteins, like PC1 and PC2, that are sorted into secretory granules and TGN-resident proteins such as furin. The 232-aa carboxyl terminus of furin is comprised of a cysteine-rich region, a membrane-spanning segment, and a hormone convertase 2; POMC, proopiomelanocortin; TGN, trans-Golgi network; vWF, von Willebrand factor; PBS, phosphate-buffered saline; CPH, carboxypeptidase H (or E); aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin
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