Elke Dittrich

A Di-leucine Motif and an Upstream Serine in the Interleukin-6 (IL-6) Signal Transducer gp130 Mediate Ligand-induced Endocytosis and Down-regulation of the IL-6 Receptor

The interleukin-6 (IL-6) receptor complex is composed of two different subunits, the IL-6 binding protein (IL-6R, gp80) and the signal transducing component gp130. Our previous studies revealed that the 10-amino acid sequence TQPLLDSEER within the intracellular domain of gp130 is crucial for the efficient internalization of IL-6. Since this sequence contains a putative di-leucine internalization motif, we further analyzed this region by constructing two additional deletions and a series of point mutants. Analyses of these mutants showed that the di-leucine pair (Leu-145 and Leu-146) is essential for ligand internalization, with leucine 145 being less resilient to exchanges. Furthermore, when a chimeric protein (Tac-STQPLL) composed of the Tac antigen fused to the hexapeptide STQPLL of gp130 was studied, we found that this sequence is sufficient to mediate endocytosis and lysosomal targeting of the chimera. Mutational analysis of three serine residues upstream of the di-leucine motif revealed that mutation of serine 139 to an alanine reduces the initial internalization rate by 50%. This finding suggests that a serine phosphorylation may be important for rapid endocytosis.

The hepatic interleukin-6 receptor Down-regulation of the interleukin-6 binding subunit (gp80) by its ligand

Interleukin‐6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6‐binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp13O). We studied the role of gp80 in binding, internalization and down‐regulation of the hepatic IL6‐receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp8O with parental cells indicate that gp80 is responsible for low affinity binding (K d = 500 pM) of IL6. Furthermore, gp80 is rate‐limiting in internalization and degradation of IL6. Internalization resulted in a rapid down‐regulation (t ≈ 15–30 min) of IL6‐binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6‐binding sites at the cell surface required >8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down‐regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.

Differential shedding of the two subunits of the interleukin‐6 receptor

cDNAs coding for the two receptor subunits of the interleukin‐6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL‐6 binding protein (IL‐6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL‐6R gp 130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL‐6R and gp 130 released by transfected cells can form a ternary complexe with interleukin‐6 indicating that such complexes also may occur in vivo. gp 130; Interleukin‐6; Interleukin‐6‐receptor; Protein kinase C; Shedding

A complex of the soluble interleukin-6 receptor and interleukin-6 is internalized via the signal transducer gp130

In human body fluids a soluble form of the interleukin‐6 receptor (sIL‐6R) has been found which together with interleukin‐6 (IL‐6) acts agonistically on cells expressing the signal transducer gp130. The means by which the sIL‐6R is removed from the circulation is unknown. Here, we show that a complex of 125I‐labelled recombinant sIL‐6R and IL‐6 is internalized by MDCK cells stably transfected with gp130 and by human hepatoma cells HepG2 that endogenously express the IL‐6R and gp130. We further show that most of the internalized sIL‐6R is degraded within lysosomes. Our studies suggest that cells expressing gp130 are capable of endocytosing an IL‐6/sIL6R complex, thereby removing both from the circulation.

Endocytosis of interleukin-6—soluble interleukin-6 receptor complex and its intralysosomal degradation

Binding and internalization of interleukin-6—soluble interleukin-6 receptor complex by MDCK and MDCK-gp130 (transfected with gp 130 signal transductor) cells are studied. Binding of labeled complex depends on the concentration of interleukin-6; an effective internalization of the complex is shown. Binding and endocytosis of the complex are demonstrated in human hepatoma cells expressing interleukin-6 receptor and gp 130. These processes depend on the concentration of interleukin-6. The inhibitors of lysosomal functions ammonium chloride, monensin, and leupeptin suppress intralysosomal degradation of the complex, which confirms the important role of intralysosomal cleavage of the complex.