Molecular modeling-guided mutagenesis of the extracellular part of gp130 leads to the identification of contact sites in the interleukin-6 (IL-6).IL-6 receptor.gp130 complex.

The transmembrane protein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal-transducing subunit. In the case of interleukin-6 (IL-6), the signal-transducing complex is composed of the ligand IL-6, the IL-6 receptor (IL-6R, gp80, CD126), and at least two gp130 (CD130) molecules. The extracellular part of the signal transducer gp130 consists of six fibronectin type III-like domains. It has recently been shown that the three membrane distal domains bind to the IL-6. IL-6R complex. A structural model of the IL-6.IL-6R.gp130 complex enabled us to propose amino acid residues in these domains of gp130 interacting with IL-6 bound to its receptor. The proposed amino acid residues located in the B'C' loop (Val252) and in the F'G' loop (Gly306, Lys307) of domain 3 and in the hinge region (Tyr218) connecting domains 2 and 3 of gp130 were mutated to disturb ternary complex formation. Binding of wild type and mutants of the extracellular region of gp130 was studied by use of a co-precipitation assay and Scatchard analysis. All mutants showed decreased binding to the IL-6.IL-6R complex. Biological function of the membrane-bound gp130 mutants was studied by STAT (signal transducer and activator of transcription) activation in COS-7 cells and by proliferation of stably transfected Ba/F3 cells. Reduced binding of the mutants was accompanied by decreased biological activity. The combined approach of molecular modeling and site-directed mutagenesis has led to the identification of amino acid residues in gp130 required for complex formation with IL-6 and its receptor.

Normal FHIT transcripts in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region.

The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer. Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer. We have analyzed the gene and its transcripts in lung cancers and renal cell cancer-derived cell lines. A lung adenocarcinoma cell line, GLC-A2, appeared to have a homozygous deletion in intron 5 of FHIT. RT-PCR analysis revealed a normal-sized PCR product in all of the cell lines: Including GLC-A2. A number of them had an additional aberrant product. Analysis of a great number of control cell lines and tissues showed that the majority of these also had aberrant PCR products in addition to a normal-sized PCR product. Different specimens of the same cell type showed variable additional RT-PCR products. Normal-sized PCR products had a sequence identical to the FHIT sequence. PCR products longer than normal had insertions of different sizes at different positions. With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons. Thus, the presence of aberrant transcripts is not cancer-specific. Conceivably, sequence responsible for the instability of the FRA3B region are being transcribed into FHIT pre-mRNA and may cause the abnormal splicing and processing of the transcripts.

A single STAT recruitment module in a chimeric cytokine receptor complex is sufficient for STAT activation.

We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor alpha and beta chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the alpha and the beta chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates. We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.

Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation.

Several cytokines and growth factors activate transcription of their target genes via the JAK/STAT signalling pathway. It has been shown that the interaction between SH2 domains of STAT factors and receptor phosphotyrosine residues plays an essential role in the specific recruitment of STATs. For STAT5, however, the importance of receptor tyrosines is still controversial. Using a chimeric receptor system in COS-7 cells, we studied the activation of STAT5 through the interleukin-6 signal transducer gp130. In contrast to previous reports, we did not detect gp130-mediated STAT5 activation. However, STAT5 activation was achieved when tyrosine motifs of other cytokine receptors were fused to the membrane-proximal part of gp130. The comparison of the relative potency of different tyrosine motifs revealed that hydrophobic amino acids, preferentially leucine, in positions +1 and +3, and an aspartate residue in position -1 or -2 with respect to the tyrosine are likely to be required for efficient STAT5 recruitment. In summary, we show here for the first time that phosphotyrosine motifs can confer the ability to activate STAT5 to a heterologous receptor.

Differential activation of acute phase response factor/Stat3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation.

Distinct yet overlapping sets of STAT transcription factors are activated by different cytokines. One example is the differential activation of acute phase response factor (APRF, also called Stat3) and Stat1 by interleukin 6 and interferon-gamma. Interleukin 6 activates both factors while, at least in human cells, interferon-gamma recruits only Stat1. Stat1 activation by interferon-gamma is mediated through a cytosolic tyrosine motif, Y440, of the interferon-gamma receptor. In an accompanying paper (Gerhartz, C., Heesel, B., Sasse, J., Hemmann, U., Landgraf, C., Schneider-Mergener, J., Horn, F., Heinrich, P. C., and Graeve, L. (1996) J. Biol. Chem. 271, 12991-12998), we demonstrated that two tyrosine motifs within the cytoplasmic part of the interleukin 6 signal transducer gp130 specifically mediate APRF activation while two others can recruit both APRF and Stat1. By expressing a series of Stat1/APRF domain swap mutants in COS-7 cells, we now determined which domains of Stat1 and APRF are involved in the specific recognition of phosphotyrosine motifs. Our data demonstrate that the SH2 domain is the sole determinant of specific STAT factor recruitment. Furthermore, the SH2 domain of Stat1 is able to recognize two unrelated types of phosphotyrosine motifs, one represented by the interferon-gamma receptor Y440DKPH peptide, and the other by two gp130 YXPQ motifs. By molecular modeling, we propose three-dimensional model structures of the Stat1 and APRF SH2 domains which allow us to explain the different binding preferences of these factors and to predict amino acids crucial for specific peptide recognition.

Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. I. Definition of a novel phosphotyrosine motif mediating STAT1 activation.

Interleukin-6 (IL-6) and gamma-interferon (IFNgamma) activate an overlapping set of genes via the Jak/STAT pathway. However, at least in human cells, a differential activation of STAT transcription factors was observed: IL-6 activates both acute phase response factor (APRF)/STAT3 and STAT1, whereas IFNgamma leads only to STAT1 activation. All STATs cloned so far contain SH2 domains. Since all cytokine receptors using the Jak/STAT pathway were found to be tyrosine-phosphorylated after ligand binding, it has been proposed that specific phosphotyrosine modules within the cytoplasmic domain of the receptor chains recruit different STAT factors. We have analyzed by mutational studies and by phosphopeptide competition assays which of the tyrosine modules of the IL-6 signal transducer gp130 are capable of recruiting either APRF or STAT1. We found that two of the four tyrosine modules that are important for APRF activation also activate STAT1. For these modules, we propose the new consensus sequence YXPQ. We further present evidence that STAT1 is activated independently from APRF suggesting that gp130 contains multiple independent STAT binding sites. We compare the APRF and STAT1 activation motifs of gp130 with the STAT1 activation motif of the IFNgamma receptor and demonstrate that the specificity of activation can be changed from APRF to STAT1 and vice versa by only two point mutations within a tyrosine module. These data strongly support the concept that the activation of a specific STAT is determined mainly by the phosphotyrosine module. The significance of these findings for other receptor systems is discussed.

Biosynthesis and half-life of the interleukin-6 receptor and its signal transducer gp130.

Interleukin-6 (IL-6) exerts its action via a receptor complex composed of a ligand-binding subunit (gp80) and a signal transducer (gp130) which both belong to the hematopoietic receptor super-family. Very little is known about the biosynthesis and the biological half-lives of proteins of this superfamily. Therefore, we studied the biosynthesis and maturation of the interleukin-6 receptor and its signaling subunit gp130 by pulse chase experiments in stably transfected Madin-Darby canine kidney cells. We found that both proteins are synthesized as precursors with apparent molecular masses of 67 kDa and 130 kDa, respectively. These receptor forms are processed within 45-60 min into mature proteins of 82 kDa and 150 kDa containing complex-type oligosaccharides. The signal transducer gp130 shows a similar maturation in human hepatoma cells HepG2. The IL-6 receptor appears at the cell surface 45 min after completion of its synthesis in the endoplasmic reticulum. In both cell types studied, gp80 and gp130 are rapidly turned over with half-lives of 2-3 h. These half-lives were unaffected by the presence of the ligand IL-6.

Identification of a region within the cytoplasmic domain of the interleukin-6 (IL-6) signal transducer gp130 important for ligand-induced endocytosis of the IL-6 receptor.

Interleukin-6 (IL-6) exerts its action via a cell surface receptor complex consisting of two subunits, the IL-6 receptor and the signal transducer gp130. We have studied the role of both transmembrane proteins for IL-6 internalization and ligand-induced down-regulation of cell surface receptors. Co-expression of wild-type and mutant forms of both the IL-6 receptor and gp130 in transiently transfected COS-7 cells revealed that gp130 is essential for efficient endocytosis and receptor down-regulation. Whereas the cytoplasmic domain of the IL-6 receptor is not significantly involved in the internalization process, deletion of the corresponding domain of gp130 resulted in an almost complete loss of the ability to endocytose IL-6. Mutants with different truncations within the intracellular domain of gp130 revealed that a 10-amino acid sequence TQPLLDSEER is crucial for efficient internalization. Since this sequence contains a putative di-leucine internalization motif, we suggest that a di-leucine motif directs the receptor mediated endocytosis of the IL-6 receptor complex.

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 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 complex with interleukin-6 indicating that such complexes also may occur in vivo.