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

IL-l-stimulated mesenchymal cells model molecular mechanisms of inflammatio n. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-sub unit signaling complex at focal adhesion complexes. Since Rho family GTPase s coordinately organize actin cytoskeleton and signaling to regulate cell p henotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved H eLa cells in a Rho-dependent manner and rapidly activated nucleotide exchan ge on RhoA. Glutathione S-transferase (GST) fusion proteins, containing eit her 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, spec ifically coprecipitated both RhoA and Rac-1, but not p21(ras), from Triton- soluble HeLa cell extracts. In whole cells, a small-molecular-weight G prot ein coimmunoprecipitated by anti-IL-1R antibody was a substrate for C3 tran sferase, which specifically ADP-ribosylates Rho GTPases. Constitutively act ivated RhoA, loaded with [gamma(-32)P]GTP, directly interacted with GST-IL- 1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functional ly important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd- directed transcriptional activation basic protein (MBP) kinases are necessa ry for IL-1-directed gene expression, cellular incorporation of C3 transfer ase inhibited IL-1R-associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically as sociated with IL-1Rcd and necessary for activation of cytosolic nuclear sig naling pathways. These findings suggest that IL-1-stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific ar chitectures that are necessary for persistent cell activation in chronic in flammatory disease.