Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage

Activation of Furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus Furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for Enzyme activation. Rather, full activation of Furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of Furin. Exposure of membrane preparations containing an inactive RER-localized soluble Furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a Furin Inhibitor. Consistent with these findings, following cleavage in the RER, the Furin propeptide remains associated with the Enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of Furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of Furin, and the relationship of the Furin activation pathway to those of other serine endoproteases are discussed.