Claudia Gerhartz

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

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.

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 α and β 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 α and the β 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.