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 dileucine 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.

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 gpl30 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [3"S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the ILdR gp130 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 gp130 released by transfected cells can form a ternary complexe with interleukin-6 indicating that such complexes also may occur in vivo.

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.

Signalling receptors often undergo receptor-mediated endocytosis. In many cases this internalization is stimulated by ligand binding and activation of intrinsic receptor tyrosine kinases, resulting in a receptor down-regulation. We have analysed whether internalization of the interleukin 6 signal transducer gp130 is dependent on the activation of receptor-associated Jak kinases. By using a chimaeric receptor system we found that receptor mutants that lack box1 and therefore are not capable of activating Jak and signal transducer and activator of transcription (STAT) proteins are still endocytosed efficiently. A chimaeric receptor with the recently identified dileucine internalization motif being replaced by two alanine residues was not efficiently internalized but still capable of recruiting STATs. Furthermore an antagonistic antibody that inhibits the signalling of all interleukin-6-type cytokines via gp130 was internalized as efficiently as an agonistic one that activates the Jak/STAT pathway. Our findings suggest that the endocytosis of gp130 is signal-independent.