The IL-1 receptor and Rho directly associate to drive cell activation in inflammation
R. Singh, … , M. Konieczkowski, J.R. Sedor
R. Singh, … , M. Konieczkowski, J.R. Sedor
Published June 1, 1999
Citation Information: J Clin Invest. 1999;103(11):1561-1570. https://doi.org/10.1172/JCI5754.
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Article

The IL-1 receptor and Rho directly associate to drive cell activation in inflammation

Abstract

IL-1–stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1–dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21ras, from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti–IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [γ-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd–directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R–associated myelin basic protein (MBP) kinases are necessary for IL-1–directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R–associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1–stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.

Authors

R. Singh, B. Wang, A. Shirvaikar, S. Khan, S. Kamat, J.R. Schelling, M. Konieczkowski, J.R. Sedor

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GST-IL-1Rcd fusion proteins. A series of GST-IL-1R fusion proteins were ...
GST-IL-1Rcd fusion proteins. A series of GST-IL-1R fusion proteins were purified from E. coli as described in Methods. (a) The full-length, 569–amino acid human IL-1R is shown for reference. The mature single-transmembrane protein is 552 amino acids. The extracellular domain (open region, ED), including a 17–amino acid signal peptide, spans the amino acids from 1 to 336; the transmembrane region (filled region, TM) from 337 to 357; and the cytosolic domain (hatched region, CD) from 358 to 569. (b) GST and GST-IL-1R fusion proteins are shown. GST-IL-1Rcd contains amino acids 369–569 of IL-1Rcd, fused to GST. The ClaI site in the IL-1Rcd cDNA sequence, used to remove the sequences encoding the COOH-terminal amino acid residues required for downstream signaling, is indicated. The resulting fusion protein, GST-IL-1RcdΔ, contains membrane-proximal cytosolic domain region amino acids 369–501 fused to GST. GST-IL-1Rkb contains the COOH-terminus signaling domain (amino acids 501–569), and its construction is described in Methods.