PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtilisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.

A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and ␣-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr 594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr 594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp 616 ), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu 583 ). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/ PC2Pd was expressed in ␣-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.Recently, a new family of serine proteases that process a wide variety of proprotein substrates has been identified in eukaryotic cells. Based on the similarity of their catalytic domain to the subtilisins, these proteases have been named subtilisin-like proprotein convertases (SPCs) 1 (Refs. 1,8,9). Each convertase has a distinct but overlapping substrate specificity and a distinctive tissue distribution, subcellular location, and maturation process, consistent with its unique role in some aspect of proprotein processing. Well characterized examples regarding these aspects are furin (expressed ubiquitously in almost all tissues), PC3, and PC2 (both restrictedly distributed in neuroendocrine tissues). We have only a limited understanding of the structural determinants which differentiate the various SPCs from each other in their function and properties. Their basic domain structure includes (Fig. 1) a signal peptide, a partially conserved propeptide, a highly conserved catalytic domain (40 -50% identity among the SPCs and 25-30% to the subtilisins) followed by a relatively well conserved region called the P, homoB, or "middle" domain. Studies in recent years have clarified the role of propeptide cleavage in furin activation and the function of the propeptide as an intramolecular inhibitor which preve...

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