Activation and routing of membrane-tethered prohormone convertases 1 and 2

Many peptide hormones and neuropeptides are processed by members of the sub tilisin-like family of prohormone convertases (PCs), which are either solub le or integral membrane proteins. PC1 and PC2 are soluble PCs that are prim arily localized to large dense core vesicles in neurons and endocrine cells . We examined whether PC1 and PC2 were active when expressed as membrane-te thered proteins, and how tethering to membranes alters the biosynthesis, en zymatic activity, and intracellular routing of these PCs. PC1 and PC2 chime ras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine cu-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to t he Golgi region. In addition, the chimeras were transiently expressed at th e cell surface and rapidly internalized to the Golgi region in a fashion si milar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro domain maturation rates, N-glycosylation, and in the pH and calcium optima requir ed for maximal enzymatic activity against a fluorogenic substrate. In addit ion, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic d omain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.