Intracellular activation of human adamalysin 19/disintegrin and metalloproteinase 19 by furin occurs via one of the two consecutive recognition sites

Adamalysin 19 (a disintegrin and metalloproteinase 19, ADAM19, or meltrin beta) is a plasma membrane metalloproteinase. Human ADAM19 zymogen contains two potential furin recognition sites (RX(K/R)R), (KRPRR)-K-196-R-200 and (RRMKR)-R-199-R-203, between its pro- and catalytic domains. Protein N-terminal sequencing revealed that the cellular mature forms of hADAM19 started at (EDLNSMK)-E-204, demonstrating that the preferred furin cleavage site was the (200)RMK(203)Rdown arrow(204)EDLN. Those mature forms were catalytically active. Both Pittsburgh mutant of alpha(1)-proteinase inhibitor and dee-Arg-Val-Lys-Arg-chloromethyl ketone, two specific furin inhibitors, blocked the activation of hADAM19. Activation of hADAM19 was also blocked by brefeldin A, which inhibits protein trafficking from the endoplasmic reticulum to the Golgi, or A23187, a calcium ionophore known to inhibit the autoactivation of furin. When (KR)-K-202 were mutated to AA, the proenzyme was also activated, suggesting that (RPRR)-R-197 is an alternative activation site. Furthermore, only pro-forms of hADAM19 were detected in the (RR)-R-199 to AA mutant, which abolished both furin recognition sites. Moreover, the zymogens were not converted into their active forms in two furin-deficient mammalian cell lines; co-expression of hADAM19 and furin in these two cell lines restored zymogen activation. Finally, co-localization between furin and hADAM19 was identified in the endoplasmic reticulum-Golgi complex and/or the trans-Golgi network. This report is the first thorough investigation of the intracellular activation of adamalysin 19, demonstrating that furin activated pro-hADAM19 in the secretory pathway via one of the two consecutive furin recognition sites.