Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)-containing Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG)

Abstract

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 10(7)-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.

FIGURE 1.

Schematic representation of domain organization and different constructs of p115, p190, PRG, and LARG used in this study. The numbers indicate the N- and C-terminal amino acids of the respective constructs. DH-PHn, DH-PHc, and DH-PHcn are shorter variants at the N and C termini of p115 DH-PH that are equivalent to LARG DH-PH (see supplemental Fig. S2A). DH-PHcnΔN and DH-PHΔN are N-terminally deleted variants of p115 and LARG and equivalent to each other. DH-PHΔN2m contains two point mutations at positions Asn⁹⁴⁶ and Lys⁹⁴⁹ that are substituted by Ser and Gln, the corresponding residues in p115. C1, cysteine-rich region; cc, coiled coil; L, leucine-rich; P, proline-rich.

FIGURE 2.

Rho specificity of p115, p190, PRG, and LARG. A and B, DH-PH catalyzes the very slow intrinsic nucleotide exchange reaction by several orders of magnitude. The mantGDP dissociation from 0.1 μm RhoA was monitored after addition of 20 μm unlabeled GDP in the absence (A) and in the presence of 2 μm LARG DH-PH (B). Note the dimension of the x axis, which is in hours in A and in seconds in B, visualizing a rate acceleration of more than 38,000-fold. C, Rho isoform specificity of p115, p190, PRG, and LARG. The observed rate constants (k_obs) of both intrinsic and DH-PH-catalyzed reactions of different GTPases were obtained by single exponential fitting of the data. The k_obs values were determined using 0.1 μm mantGDP-bound GTPases (RhoA, RhoB, RhoC, Rac1, Cdc42, and TC10) and 20 μm non-fluorescent GDP in the mantGDP dissociation catalyzed by four different DH-PH proteins (2 μm each). D, DH-PH-catalyzed nucleotide exchange is independent of the type of bound nucleotide. GEF-catalyzed mantGDP and mantGppNHp dissociation from RhoA was monitored using 0.1 μm mant-nucleotide-loaded RhoA (RhoA-mantGDP or RhoA-mantGppNHp) and 20 μm non-fluorescent nucleotide (GDP or GppNHp) in the presence of 10 μm DH-PH domain of p115 or p190 or 2 μm DH-PH domain of PRG or LARG. Note that a 5-fold lower concentration of LARG and PRG has been used compared with the experiments with p190 and p115. Moreover, the LARG-catalyzed mant-nucleotide dissociation was measured in the presence of excess amounts of both GDP and GppNHp (white bar). The observed rate constants (k_obs) were obtained by single exponential fitting of the data. For convenience, the exact k_obs values are given as numbers above the bars in C and D.

FIGURE 3.

Kinetics of catalyzed nucleotide dissociation reaction of RhoA by RhoGEFs PRG, p190, p115, and LARG using fluorescent nucleotides. A, kinetics of mantGDP dissociation from RhoA (0.1 μm) were measured in the presence of 20 μm non-fluorescent GDP and increasing concentrations of the DH-PH domain of PRG (1, 2, 5, 10, 20, and 50 μm) under the same condition as in Fig. 2. Observed rate constants (k_obs) of the respective data were obtained by single exponential fitting. The dependence of the k_obs values for the mantGDP dissociation on the concentrations of the DH-PH (closed circles) and the DH (open circles) domains of PRG (B), p190 (C), p115 (D), and LARG (E) was fitted to a hyperbolic curve to obtain the kinetic parameters of the GEF-catalyzed nucleotide dissociation from RhoA.

FIGURE 4.

Real time monitoring of RhoGEF interactions with GDP-bound fRhoA. A, RhoA labeling strategy with the fluorescence reporter group AEDANS (inset). The van der Waals surface of nucleotide-free RhoA from the LARG DH-PH complex (17) (Protein Data Bank code 1X86) shows the solvent-accessible surrounding residues (green) around the interaction surface of LARG (orange). Valine 33 (V33) of RhoA substituted by cysteine and labeled with AEDANS (fRhoA) is shown in red. B, fRhoA allows monitoring of the RhoGEF association in real time. Rapid mixing of increasing p115 DH-PH concentrations (0.5–5 μm) with fRhoA-GDP (0.2 μm) resulted in an incremental increase in fluorescence corresponding to the association reaction. C, the association rate constants (k_on) of fRhoA-GDP binding to the DH and DH-PH proteins of LARG and p115, respectively, clearly revealed differences in the binding properties of the two RhoGEFs. D, the dissociation rate constant (k_off) of the DH and DH-PH proteins of LARG and p115, respectively, displaced from the fRhoA-GDP complex in the presence of excess amounts of unlabeled, nucleotide-free RhoA revealed an impact of p115 PH domain on the GEF dissociation kinetics. The kinetic data are shown in supplemental Fig. S3. The dissociation constant (K_d) was calculated from the kinetic parameters of dissociation and association reactions by the equation K_d = k_off/k_on. For convenience, the exact k_on and k_off values are given as numbers above the bars in C and D, respectively.

FIGURE 5.

Critical role of N-terminal segment of DH domain in association and nucleotide exchange reactions. A, possible new signatures for the DH function. The crystal structure (17) (Protein Data Bank code 1X86) of the nucleotide-free RhoA (violet) in the complex with LARG DH-PH (turquoise) highlights eight residues (orange) in the DH domain that may be critical for the efficiency of LARG in both associating with GDP-bound RhoA and catalyzing nucleotide dissociation. Four of the eight residues are located in a short peptide called the N-terminal (N-term.) segment (green). Switch (Sw) regions I and II of RhoA and shown in blue and red, respectively. B, the k_obs values highlight the association efficiency of the DH-PH variants of LARG and p115 (5 μm, respectively) with 0.2 μm fRhoA-GDP. C, the k_obs values show the exchange reaction of mantGDP from RhoA (0.1 μm) catalyzed by the DH-PH variants of LARG and p115 (10 μm, respectively). For convenience, the exact k_obs values are given as numbers above the bars in B and C.

FIGURE 6.

RGS-Linker-mediated autoinhibition of p115 DH Activity. The effects of various p115 domains on the intrinsic and the DH-catalyzed mantGDP dissociation from RhoA were measured under the same conditions as in Fig. 2. The following protein concentrations were used: 0.1 μm RhoA-mantGDP, 1 μm DH, 1 μm RGS-Linker-DH, 10 μm RGS, and 10 μm Linker. The observed rate constants (k_obs) of both intrinsic and catalyzed reactions were obtained by single exponential fitting of the data. For convenience, the exact k_obs values are given as numbers above the bars.

FIGURE 7.

Structure-based interaction sequence matrix illustrating specificity determining residues for RhoA interaction with its GEFs. Based on the crystal structures of RhoA (G domain) in the complex with DH-PH of PRG (16) (Protein Data Bank code 1XCG) and of LARG (17) (Protein Data Bank code 1X86) the interacting residues (colored background; <4 Å in distance) were determined and aligned onto the DH-PH tandem and the G domain of RhoGTPases. Residues with a light blue background are conserved in Rho-specific GEFs and critical in determining the specificity of the RhoA/DH-PH interaction. Variable residues with a black background may be critical in determining the catalytic efficiency of Rho-specific GEFs.