Identification of a Transferable Sorting Domain for the Regulated Pathway in the Prohormone Convertase PC2

doi:10.1074/jbc.271.41.25284

Identification of a Transferable Sorting Domain for the Regulated Pathway in the Prohormone Convertase PC2*

From the ^‡ Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom and the
^″ Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium

^¶ Previously a Human Capital and Mobility Fellow of the European Union. To whom correspondence should be addressed. Present address:
Center for Human Genetics, University of Leuven, Herestraat 49, *B-3000 Leuven, Belgium
. Tel.: 32-16-346080; Fax: 32-16-346073.

↵^‴ Present address: Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Box B140, 4200 East 9th Ave., Denver, CO 80262.

↵^§ Both authors contributed equally.

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.

Footnotes

↵^∥ Previously a holder of a predoctoral fellowship from Fundació Clinic, Barcelona and from Direcció General de Recerca CIRIT, Generalitat de Catalunya. Current address: Servei d'Endocrinologia i Nutrició, Secció Diabetis, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain.
↵* This work was supported by the Wellcome Trust, British Diabetic Association, the Medical Research Council of Great Britain, the “Geconcerteerde Onderzoeksacties 1992-1996,” and the “Stichting Technische Wetenschappen” (STW-22.2726). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

PC2

prohormone 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.
↵² E. M. Bailyes, personal communication.
- Received May 23, 1996.
- Revision received July 15, 1996.