Activation of leukemia-associated RhoGEF by Galpha13 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 Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(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 Galpha(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 Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(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 Galpha(13).

FIGURE 1.

Direct interaction of LARG with Gα₁₃ through its RH domain, DH/PH domains, and C-terminal region. A, schematic representation of LARG and its deletion constructs. The amino acid numbers encoded in each constructs are listed. PDZ, PDZ domain; RH, RGS homology domain; DH, Dbl homology domain; PH, pleckstrin homology domain. A full-length, RDPC, DPC, RDP, PDZ, RH, DH/PH, or C-terminal region of LARG is referred to as LARG-FL, -RDPC, -DPC, -RDP, -PDZ, -RH, -DH/PH, or -C, respectively. B, the binding of various LARG proteins to Gα₁₃ in COS1 cells. COS1 cells were co-transfected with Gα₁₃WT (0.5 μg) and the indicated myc-tagged LARG constructs: RDPC (5 μg), DPC (4 μg), DH/PH (3 μg), C(4 μg), and RH (5 μg). The LARG proteins were immunoprecipitated by anti-Myc antibody from cell lysates in the presence or absence of AMF. Immunoprecipitates were separated by SDS-PAGE, followed by Western blotting using anti-Gα₁₃ antibody or anti-Myc antibody. C, kinetics of binding of LARG to Gα_i/13 or Gα_i/13KA immobilized on the SPR biosensor. Gα_i/13 and Gα_i/13KA proteins were immobilized on parallel channels of the Biacore sensor chip CM5 as described under “Experimental Procedures.” The association phase of the reaction between serially diluted LARG fragments and Gα_i/13 was 2 min, and the dissociation phase was 1 min at 15 °C. The interactions were measured using Biacore 3000. Black lines show the experimental data. Red lines show fitting data analyzed as simultaneous k_a/k_d, 1:1 binding, and global fitting using the BIAevaluation program. In the absence of AMF, kinetic analyses were not performed when the responses were <1/10 of those with Gα_i/13 in the presence of AMF. The concentrations of proteins were: FL, 1.1–17.5 nm; RDPC, 4.4–70 nm; RDP, 1.1–17.5 nm; DPC, 37.5–600 nm; RH, 8.8–140 nm; and DH/PH, 78.1 nm to 1.25 μm.

FIGURE 2.

RhoGEF activation of LARG by the direct interaction of the DH/PH domains with Gα₁₃. A, potentiation of SRF activation by LARG with Gα₁₃. HeLa cells were cotransfected with 0.1 μg of SRE-luciferase reporter plasmid and the indicated constructs: Gα₁₃QL (0.01 μg), myc-LARG-RDPC (0.1 μg), myc-LARG-DPC (0.1 μg), myc-LARG-DH/PH (0.01 μg), and myc-LARG-C (0.1 μg). SRF activities of cell lysates were measured 24 h after transfection as described in the supplemental materials. B, RhoA activation by LARG-DH/PH with Gα₁₃ in HeLa cells. HeLa cells were transiently transfected with the indicated plasmids: Gα₁₃QL (1 μg), myc-tagged RDPC (10 μg), myc-DPC (1 μg), and myc-DH/PH (0.5 μg). The cells were serum-starved for 24 h after 5 h of transfection. Endogenous RhoA in the GTP-bound form was isolated using GST-Rhotekin RBD from the cell lysates as described in the supplemental materials. The expression of Myc-tagged LARG constructs and Gα₁₃ were analyzed by Western blotting using anti-Myc and anti-Gα₁₃ antibodies. C, in vitro RhoGEF assays of LARG-DH/PH and -RDPC. GDP dissociation from RhoA was measured at 20 °C after 2-min incubation in the presence of the indicated proteins: LARG-RDPC (20 nm), LARG-DH/PH (30 nm), -activated Gα₁₃WT (100 nm). (Student's t test: n = 4, ^*, p < 0.001). D, recruitment of LARG with RH domain to the plasma membrane by constitutively active Gα₁₃. HeLa cells were cotransfected in the same procedure as in Fig. 2A. The expression of LARG constructs in total cell lysate (T), crude cytosolic (C), and membrane (M) fractions, and Gα₁₃ in total lysates was detected by immunoblotting using anti-Myc antibody or anti-Gα₁₃ antibody. E, cell rounding induced by LARG constructs with the constitutively active Gα₁₃ in MDCKII cells. MDCKII cells were transiently transfected with the indicated myc-tagged LARG constructs in the presence or absence of Gα₁₃QL, or FLAG-tagged V14RhoA alone. The cells were serum-starved for 24 h after 5 h of transfection, then fixed, and triply stained with anti-Myc antibody, anti-Gα₁₃ antibody, and phalloidin for filamentous actin. Transfected cells were visualized by fluorescence microscopy, identified, and scored for rounding indicating the involvement of RhoA activation as described under the supplemental materials. The values were calculated from four independent experiments. The images are in supplemental Fig. S4. F, stimulation of the RhoGEF activity of LARG by Gα_i/13KA. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the following proteins: ○, control; ▵, -activated Gα_i/13 (100 nm); □, AMF-activated Gα_i/13KA (100 nm); •, RDPC (10 nm); ▴, RDPC (10 nm) plus AMF-activated Gα_i/13 (100 nm); ⋄, RDPC (10 nm) plus AMF-activated Gα_i/13KA (100 nm).

FIGURE 3.

Functional roles of the RH domain and C-terminal region for DH/PH domains-mediated GEF activation of LARG. A, Enhancement of the Gα₁₃-stimulated RhoGEF activity of LARG-DH/PH by LARG-RH in HeLa cells. HeLa cells were transiently transfected with the indicated plasmids: Gα₁₃QL (1 μg), myc-tagged LARG-DH/PH (1.5 μg), myc-LARG-RH (2 μg), or myc-p115-RH (0.02 μg). GTP-bound form of RhoA was isolated as described in Fig. 2B. The left of the panel is a representative result from three independent experiments. The right panel shows the mean ± S.E. of values of amount of GTP-RhoA scanned using ImageJ program. B, potentiation of the Gα₁₃-induced RhoGEF activity of LARG-DH/PH by LARG-RH in vitro. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the indicated proteins: AMF-activated Gα₁₃ (100 nm), RH (600 nm), DH/PH (120 nm). (Fisher's protected least significant difference test: n = 3, ^*, p < 0.001). C, enhancement of the binding of LARG-RH and LARG-DPC with Gα₁₃ immobilized on Ni-NTA-agarose. Equal amounts of cell lysates of COS1 cells transfected with myc-LARG-DPC (8 μg) or myc-LARG-RH (4 μg) were incubated with 1.4 μg of immobilized His-Gα_i/13 or His-Gα_i/13KA proteins onto Ni-NTA agarose beads in the presence or absence of AMF. The relative amount of LARG fragments bound to the activated- or deactivated-Gα₁₃ was visualized by Western blot analysis using anti-Myc antibody. The expression of the LARG proteins in total cell lysates and the Gα₁₃ protein in the reaction mixtures are also shown. D, the role of LARG C terminus in RhoGEF activation of LARG by Gα₁₃. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the indicated proteins: AMF-activated Gα₁₃ (100 nm), RDP (20 nm), C (200 nm), and RDPC (20 nm) (Student's t test: n = 3, ^*, p = 0.0001).

FIGURE 4.

Thermodynamic analysis of Gα₁₃-LARG interaction. A, thermodynamic analysis of the Gα₁₃-LARG complex formation and dissociation through its RH and DH domain. van't Hoff plots and Eyring plots of the experimental data are shown. The thermodynamic parameters at an equilibrium state and at a transition state were estimated from van't Hoff plots and Eyring plots as described under “Experimental Procedures” (Table 2 and supplemental Fig. S3). B, schematic reaction profile of the thermodynamic energies at the different states of Gα₁₃-LARG interaction.

FIGURE 5.

A proposed model of Rho activation through Gα₁₃-LARG interaction. The interaction of LARG-RH with Gα₁₃ activated by agonist-bound GPCR will induce conformational changes of DH/PH domains and C-terminal region to form an active Gα₁₃-LARG complex.