Accurate and efficient cleavage of the human insulin proreceptor by the human proprotein-processing protease furin. Characterization and kinetic parameters using the purified, secreted soluble protease expressed by a recombinant baculovirus.
Maturation of the insulin proreceptor in a late Golgi compartment requires cleavage at an Arg-Lys-Arg-Arg processing site, suggesting involvement of furin, a transmembrane serine protease of the Kex2 family of processing enzymes. A genetically engineered secreted, soluble form of human furin (ss-furin), expressed by infection of insect cells with a recombinant baculovirus, was purified to near homogeneity. ss-Furin exhibited rapid and efficient cleavage of both isoforms of the human insulin proreceptor in solubilized extracts of cultured mammalian cells expressing preproreceptor cDNA. Proreceptor cleavage occurred at the physiological processing site as judged by the effects of mutations in this site on cleavage by purified ss-furin. Moreover, purified ss-furin exhibited specificity for proreceptor cleavage identical to that of the endogenous insulin proreceptor-processing enzyme. Furin thus displays the properties expected of an insulin proreceptor-processing enzyme in that it (i) cleaves the proreceptor efficiently and at the correct site; (ii) exhibits the same specificity in processing variant proreceptors as the endogenous enzyme; (iii) appears to be localized in the correct secretory compartment; and (iv) has the same broad pattern of tissue distribution as the insulin proreceptor.
Bravo
D A
DA
Department of Biochemistry, Stanford University School of Medicine, California 94305.
Gleason
J B
JB
Sanchez
R I
RI
Roth
R A
RA
Fuller
R S
RS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D006224:Cricetinae; D045683:Furin; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D003374:Coumarins; D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C051512:insulin proreceptor; C089959:tert-butoxycarbonyl-arginyl-valyl-arginyl-arginyl-methylcoumarin amide; D011972:Receptor, Insulin; D013381:Subtilisins; D045683:Furin; D002118:Calcium