The ligand-binding subunit (gp80) of the human interleukin-6 receptor (IL-6R) was transiently expressed in COS-7 cells. The metabolically labeled protein was shown to be quantitatively released from the membrane within 20 h. We identified the protein released from the transfected COS-7 cells after purification to homogeneity and N-terminal sequencing as a soluble form of the gp80/IL-6R. Shedding of the gp80 protein was strongly induced by 4 beta-phorbol-12-myristate-13-acetate, indicating that the process was regulated by protein kinase C (PKC). This was further corroborated by the finding that co-transfection of a PKC expression plasmid led to enhanced shedding of the gp80 protein. Since shedding of gp80 could not be prevented by treatment of the cells with inhibitors of all known classes of proteases, a novel protease seems to be involved. As a control, an unrelated membrane protein (vesicular stomatitis virus glycoprotein) was transfected into COS-7 cells and analyzed for shedding. Since the turnover of this protein was not mediated by shedding, we conclude that the release of gp80 from COS-7 cells is a specific process. The shed gp80 protein specifically binds IL-6, and this complex shows biological activity on human hepatoma cells. Human peripheral blood monocytes released a soluble form of the gp80 protein into the culture medium upon PMA treatment indicating that PKC-regulated shedding is the physiological mechanism of generation of the soluble IL-6R.

lnterleukin-6 (IL6) exerts its action via a cell surface receptor composed or an 80 kDa IL6-binding protein (gp80) and a 130 kDa polypeptlde involved in signal transduetion (gp130). We studied the role of gpB0 in binding, internalization and down-regulation of the hepatic IL6-receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of tranffected HepG2 cells overexpr~sinB gpB0 with parental cell~ indicate that gpS0 is responsible for low affinity binding (Ka = 500 pM) of IL6. Furthermore, gpS0 is rate-limiting in internalization and degradation of IL6. Internalization resulted in a rapid down-regulation (t~. ~ 15-30 rain) of I L6-binding sites at the cell surface. More than B0% of the internalized [~2~l]rhlL6 was degraded. The reappearance of IL6-binding sites at the cell surface required >8 h and was sensitive to cyeloheximide0 su$Sestin B that gpB0 is not recycled after internalization. The down-regulation of the hepatic IL6R by its tigand might play an important role as a protection a~inst overstimulation.

No abstract

The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in v i m folding procedure using L-arginine and the glutathione-redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column.The functional features of the recombinant soluble receptor were further analysed.

Affinity cross-linking of '251-labeled recombinant human interleukin-6 (IL-6) to human hepatoma cells (HepG2) allowed the detection of three IL-6-containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa.Treatment of HepG2 cells with dexamethasone led to a time-and dose-dependent up-regulation of IL-6-receptor mRNA levels. By the use of cross-linking this effect was also seen at the protein level, where all three 1L-6-binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL-6-receptor up-regulation by dexamethasone, y-fibrinogen mRNA induction by IL-6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL-6 receptor might be a ratelimiting step in acute-phase-protein induction.