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.

Evidence for a protein-trafficking gene that rescues the defective glucocorticoid-regulated transport and Golgi retention of mouse mammary tumor virus glycoproteins in a rat hepatoma cell-sorting variant.

Glucocorticoids regulate the trafficking of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the virus-infected rat hepatoma cell line M1.54. The CR4 rat hepatoma sorting variant, which is derived from M1.54 cells by immunoselection, is uniquely defective in the glucocorticoid-regulated transport of MMTV glycoproteins. Indirect immunofluorescence of fixed permeabilized cells and subcellular fractionation of isolated microsomes revealed that variant CR4 cells retain the MMTV glycoproteins in Golgi-like membranes after glucocorticoid treatment. The variant CR4 phenotype can be complemented by interspecies cell fusions with human HepG2 hepatoma cells and by DNA rescue with genomic fragments isolated from either human or rat hepatoma cells. Transfected wild-type genomic fragments rescue the sorting defect in CR4 at a frequency consistant with a single genetic locus, whereas homologous transfection with CR4 genomic DNA has no effect. Thus, complementation of a rat hepatoma cell-sorting variant supports the existence of a novel protein-trafficking activity encoded by the human or rat genomes that acts in trans in the Golgi to selectively mediate the sorting of cell surface MMTV glycoproteins in glucocorticoid-treated cells.

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells.

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

Glucocorticoid responsiveness of mouse mammary tumor virus (MMTV) promoters in a down-transcription hepatoma tissue culture (HTC) variant.

Complement-mediated cytolysis of the mouse mammary tumor virus (MMTV)-infected rat hepatoma (HTC) cell line, M1.54, resulted in recovery of a mutant derivative, designated CR5, in which the magnitude of both basal and dexamethasone-induced proviral MMTV RNA expression was selectively reduced. Variant CR5 cells were transfected with a plasmid containing the glucocorticoid-regulated MMTV promoter linked to the neomycin resistance gene (pLNL). Half-maximal resistance to G418 killing was glucocorticoid inducible in both pLNL-transfected CR5 and M1.54 cells and was dependent on glucocorticoid receptor occupancy. The down-transcription of MMTV provirus sequences cannot be conferred to transfected genes driven by the same viral promoter suggesting that CR5 cells are defective in cis acting factors. Consistent with this notion, indirect immunofluorescence of transient heterokaryons revealed that uninfected wild-type HTC cells failed to complement the defect in CR5 while CR5 cells did not suppress the wild-type phenotype of M1.54 cells.

Glucocorticoid-regulated localization of cell surface glycoproteins in rat hepatoma cells is mediated within the Golgi complex.

Glucocorticoid hormones regulate the post-translational maturation and sorting of cell surface and extracellular mouse mammary tumor virus (MMTV) glycoproteins in M1.54 cells, a stably infected rat hepatoma cell line. Exposure to monensin significantly reduced the proteolytic maturation and externalization of viral glycoproteins resulting in a stable cellular accumulation of a single 70,000-Mr glycosylated polyprotein (designated gp70). Cell surface- and intracellular-specific immunoprecipitations of monensin-treated cells revealed that gp70 can be localized to the cell surface only in the presence of 1 microM dexamethasone, while in uninduced cells gp70 is irreversibly sequestered in an intracellular compartment. Analysis of oligosaccharide processing kinetics demonstrated that gp70 acquired resistance to endoglycosidase H with a half-time of 65 min in the presence or absence of hormone. In contrast, gp70 was inefficiently galactosylated after a 60-min lag in uninduced cells while rapidly acquiring this carbohydrate modification in the presence of dexamethasone. Furthermore, in the absence or presence of monensin, MMTV glycoproteins failed to be galactosylated in hormone-induced CR4 cells, a complement-selected sorting variant defective in the glucocorticoid-regulated compartmentalization of viral glycoproteins to the cell surface. Since dexamethasone had no apparent global effects on organelle morphology or production of total cell surface-galactosylated species, we conclude that glucocorticoids induce the localization of cell surface MMTV glycoproteins by regulating a highly selective step within the Golgi apparatus after the acquisition of endoglycosidase H-resistant oligosaccharide side chains but before or at the site of galactose attachment.

Glucocorticoid-dependent complementation of a hepatoma cell variant defective in viral glycoprotein sorting.

We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.