title: Activation and routing of membrane-tethered prohormone convertases 1 and 2.
abstract: Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.
authors.affiliation: Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bruzzaniti
authors.forename: R
authors.initials: R
authors.lastname: Marx
authors.forename: R E
authors.initials: RE
authors.lastname: Mains
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D016259:Genes, myc; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases; D002118:Calcium