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

Abstract

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 G␣ 13 . However, the molecular mechanism of LARG activation upon G␣ 13 binding is not yet well understood. In this study, we analyzed the G␣ 13 -LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active G␣ 13 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 G␣ 13 resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of G␣ 13 proceeds with a large negative heat capacity change (⌬Cp°), accompanied by a positive entropy change (⌬S°). These results likely indicate that the binding of G␣ 13 with the RH domain triggers conformational rearrangements between G␣ 13 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 G␣ 13 .Heterotrimeric G proteins 3 serve as key molecular switches to transduce a large array of extracellular signals into cells by actively alternating their conformations between GDP-bound inactive and GTP-bound active forms. In the current model, the ligand-activated G protein-coupled receptors (GPCRs) catalyze the exchange of GDP for GTP on G␣ subunits (1). Upon activation, three switch regions in the G␣ subunit undergo significant conformational changes, followed by dissociation of the GTP-bound G␣ subunit from the G␤␥ subunits. Both G␣-GTP and free G␤␥ interact with diverse downstream effectors to transmit intracellular signals. The G␣ subunit hydrolyzes bound GTP to GDP by its intrinsic GTPase activity. This deactivation process is further accelerated by GTPase-activating proteins (GAPs) such as regulator of G protein signaling (RGS) proteins (2, 3). G␣-GDP dissociates from effectors and re-associates with G␤␥ to terminate the signal.Although this model explains the basic concept of G protein signaling, the molecular dynamics of interactions among GPCR, G protein, RGS protein, and effector during the signaling process is not well understood. It has been suggested that the GPCR signals are integrated into the intracellular signaling network at the level of G proteins (4). Accumulating evidence suggests that the G␣ subunit acts as the core of the signaling complex at the membrane, which is formed through the transient protein-protein interactions of multiple signaling compo...

Structure and Function of Heterotrimeric G Protein-Regulated Rho Guanine Nucleotide Exchange Factors

“…3a). Using surface plasmon resonance, the first study showed that G␣ 13 could interact not only with the RH but also with the DH/PH and C-terminal regions of LARG (Suzuki et al, 2009b), consistent with earlier observations using coimmunoprecipitation (Wells et , 2003). The presence of all three domains (RH, DH, and PH) was required for formation of the highest affinity complex, and the K204A mutation of G␣ 13 , which abrogates binding to the RH domain (Nakamura et al, 2004;Grabocka and Wedegaertner, 2005), did not eliminate binding to the DH/PH domain.…”

“…Although membrane recruitment is one aspect of signal transduction to RH-RhoGEFs, additional interactions at the cell membrane involving the various structural domains and sequence motifs of the RH-RhoGEF are probably required for full activation (Bhattacharyya and Wedegaertner, 2003;Bhattacharyya et al, 2009). Indeed, there is evidence that G␣ 13 interacts with other regions in addition to the RH domain (indicated by red arrows) to stabilize a high-affinity complex and stimulate GEF activity (Suzuki et al, 2009b). Autoinhibition of basal activity is believed to be imposed by the cooperative actions of the N-terminal region and C-terminal tail of the RhoGEF, as well as by an acidic sequence in the RH-DH linker region .…”

“…There is considerable evidence that the mechanism of activation is also complex, involving not only recruitment to specific sites at the membrane surface, but also multiple intra-and intermolecular interactions that mediate inhibition of basal activity as well as allosteric activation (Bhattacharyya and Wedegaertner, 2003;Bhattacharyya et al, 2009;Suzuki et al, 2009b;Zheng et al, 2009). The specific details vary among subfamily members (e.g., p115RhoGEF lacks a PDZ domain) and context .…”

Activation of p115-RhoGEF Requires Direct Association of Gα13 and the Dbl Homology Domain

“…The binding affinities between RH and G␣ 13 range between 10 nM to 1 M, depending on the specific RH domain and the state of the GTPase. 4 Binding of G␣ 13 to regions outside of the RH domain has been suggested (9,17), but the evidence is limited. The affinity between truncated p115 missing RH and activated G␣ 13 is low, such that no physical association between the two could be observed by size exclusion chromatography (Fig.…”

“…This raises the question of whether direct association between RH and G␣ 13 is actually required for the stimulation of GEF activity; it also suggests that regions outside of RH in p115 might interact with G␣ 13 during the activation process. There is evidence that activated G␣ 13 binds weakly to regions outside of RH; however, it has not been shown that the GEF activity of p115 is regulated by such interactions (9,17).…”