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The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.
Signal transducer and activator of transcription 6 (STAT6) is a transcription factor that is activated by interleukin-4 (IL-4)-induced tyrosine phosphorylation and mediates most of the IL-4-induced gene expression. Transcriptional activation by STAT6 requires the interaction with coactivators like p300 and the CREB-binding protein (CBP). In this study we have investigated the function of the CBP-associated members of the p160/steroid receptor coactivator family in the transcriptional activation by STAT6. We found that only one of them, NCoA-1, acts as a coactivator for STAT6 and interacts directly with the transactivation domain of STAT6. The N-terminal part of NCoA-1 interacts with the far C-terminal part of the STAT6 transactivation domain but does not interact with the other members of the STAT family. This domain of NCoA-1 has a strong inhibitory effect on STAT6-mediated transactivation when overexpressed in cells, illustrating the importance of NCoA-1 for STAT6-mediated transactivation. In addition, we showed that both coactivators CBP and NCoA-1 bind independently to specific regions within the STAT6 transactivation domain. Our results suggest that multiple contacts between NCoA-1, CBP, and STAT6 are required for transcriptional activation. These findings provide new mechanistic insights into how STAT6 can recruit coactivators required for IL-4-dependent transactivation.