Activation of Leukemia-associated RhoGEF by Gα13 with Significant Conformational Rearrangements in the Interface

• Published in: The Journal of biological chemistry Vol. 284; no. 8; pp. 5000 - 5009
• Main Authors: Suzuki, Nobuchika, Tsumoto, Kouhei, Hajicek, Nicole, Daigo, Kenji, Tokita, Reiko, Minami, Shiro, Kodama, Tatsuhiko, Hamakubo, Takao, Kozasa, Tohru
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 20-02-2009; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804073200

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Gα13. However, the molecular mechanism of LARG activation upon Gα13 binding is not yet well understood. In this study, we analyzed the Gα13-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Gα13 interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Gα13 resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Gα13 proceeds with a large negative heat capacity change (ΔCp°), accompanied by a positive entropy change (ΔS°). These results likely indicate that the binding of Gα13 with the RH domain triggers conformational rearrangements between Gα13 and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Gα13.