COOH-TERMINAL PROTEOLYTIC PROCESSING OF SECRETED AND MEMBRANE FORMS OF THE ALPHA-SUBUNIT OF THE METALLOPROTEASE MEPRIN-A - REQUIREMENT OF THE I-DOMAIN FOR PROCESSING IN THE ENDOPLASMIC-RETICULUM

Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats co ntain beta subunits that are type I integral membrane proteins and alp ha subunits that are disulfide linked to or noncovalently associated w ith membrane-anchored meprin subunits. Both alpha and beta subunits ar e synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are ret ained in beta subunits but are removed from alpha during maturation. T he present studies establish that an inserted 56-amino acid domain (th e ''I'' domain), present in alpha but not in beta, is necessary and su fficient for COOH-terminal proteolytic processing of the alpha subunit . This was demonstrated by expression of mutant meprin subunits (delet ion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing site s present in the I domain (a dibasic site and a furin site) did not pr event COOH-terminal proteolytic processing, indicating that the protea ses responsible for cleavage are distinct from those having these spec ificities, Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furth ermore, COOH-terminal proteolytic processing of wild-type alpha subuni ts occurred before acquisition of endoglycosidase H resistance. Pulse chase experiments and expression of an alpha subunit transcript contai ning a c-myc epitope tag, confirmed that proteolytic lytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work id entifies the region of the alpha subunit that is essential for COOH-te rminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a str ucturally unique tetrameric protease begins in the endoplasmic reticul um.