Structural basis of RhoA activation by leukemia-associated RhoGEF
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Small GTPases of the Rho family are regulators of cytoskeletal organization, neuronal morphogenesis, and transcription and are implicated in the development of cancer. The activity of Rho GTPases is dependent on their binding to GDP (inactive) or GTP (active) and is tightly regulated by accessory proteins. RhoGEFs (Rho guanine nucleotide exchange factors) activate Rho GTPases by stabilizing their nucleotide-free form via a DH/PH (Dbl homology/Pleckstrin homology) domains module. Leukemiaassociated RhoGEF (LARG) belongs to a subfamily of RhoGEFs, the RH-RhoGEFs, that also contain an RH (Regulator of G protein signaling homology) domain N-terminal to the DH/PH domains and specifically activate RhoA, and not the two other RhoGTPases, Cdc42 and Rac1. RH-RhoGEFs are coupled to G protein-coupled receptor (GPCR) activation because their RH domains interact with Gα12/13 proteins, which in turn activates their GEF activity. Although the LARG DH domain is sufficient for catalysis the PH domain contributes to nucleotide exchange. To better understand how the LARG PH and RH domains contribute to its activity, and to elucidate the structural determinants of RhoA-specificity, structures of the LARG DH/PH domains alone and in complex with RhoA were determined by x-ray crystallography at 2.1 and 3.2 Å resolution, respectively. To verify the structural findings, mutants were generated and assessed using a fluorescence assay. A novel N-terminal subdomain of the DH domain was discovered, which seems to be important for activity and might be used as a switch to regulate LARG activity. The sequence of this N-terminal extension is conserved throughout the Lbc family of RhoA specific RhoGEFs. PH domain-assisted nucleotide exchange in LARG is dependent on the structural integrity of the junction between the DH and PH domains and not on direct contacts of the PH domain with RhoA. A hydrophobic patch on the PH domain has been discovered, which might be a protein-docking site that is used for regulation of GEF activity by other proteins (e.g Gα13) or other domains within LARG (e.g. RH domain). Fluorescence assays of fragments including the RH domain showed an inhibitory effect of the RH domain in vitro, the structural basis of which will be investigated in future experiments.