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

Abstract

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.

When cell biology meets development: endocytic regulation of signaling pathways

“…These internalization-sorting signals may be modified by posttranslational modification, which may serve to regulate both constitutive and ligand-activated endocytosis. Phosphorylation of residues within or adjacent to the internalization motifs has been shown to affect receptor internalization, and may be responsible for the increased rate of internalization observed after ligand binding (Dietrich et al 1994;Dittrich et al 1996;Pitcher et al 1999). In addition, endocytic motifs can be ubiquitinated.…”

Interleukin‐6: Structure‐function relationships

“…After binding to the ILdR, IL-6 is rapidly internalized -15-30 min) leading to receptor downregulation Zohlnhofer et al, 1992). A region spanning amino acids 142-151 (TQPLLDSEER) and an upstream serine (Ser 139) in the cytoplasmic domain of gp130 were identified as being crucial for E-6 internalization and downregulation of the K-6R (Dittrich et al, 1994(Dittrich et al, , 1996. The presence in this region of a di-leucine motif, which has also been described as a lysosomal targeting signal (for a review see Trowbridge et al, 1993), suggests that the gpl30-related receptors are endocytosed via clathrin-coated pits.…”

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1

“…After p27 detection blots were stripped and hybridized with a GAPDH cDNA probe. Fold increase of p27 mRNA normalized according to GAPDH values was calculated using the ImageQuant software after scanning of the blots with a PhosphorImager (Molecular Dynamics) of gp130 (Dittrich et al, 1996;Thiel et al, 1998) and which may account for the lower surface expression of the full-length EG construct compared to the shorter constructs (Figure 3). Studies are in progress to answer this question.…”