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)

Mamta Jaiswal,Lothar Gremer,Radovan Dvorsky,Lars Christian Haeusler,Ion C Cirstea,Katharina Uhlenbrock,Mohammad Reza Ahmadian

doi:10.1074/jbc.m111.226431

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.

Highlights

The small GDP/GTP-binding proteins (GTPases)4 of the Rho family are key regulators in a multitude of cellular processes [1, 2]
Dbl homology (DH)-pleckstrin homology (PH) Tandem Determines guanine nucleotide exchange factors (GEFs) Specificity—Considerable advantages in the investigation of the GEF-accelerated nucleotide exchange reaction are provided by fluorescence spectroscopy [38, 41, 43]
The very slow intrinsic nucleotide dissociation rate (1.7 ϫ 10Ϫ5 sϪ1) was efficiently accelerated 3.8 ϫ 104-fold when 2 ␮M of the DH-PH domain of leukemia-associated RhoGEF (LARG) was mixed in the stopped-flow apparatus with 0.1 ␮M RhoA-mantGDP and 20 ␮M GDP (Fig. 2B)

Summary

Introduction

The small GDP/GTP-binding proteins (GTPases)4 of the Rho family are key regulators in a multitude of cellular processes [1, 2]. The very slow intrinsic nucleotide dissociation rate (1.7 ϫ 10Ϫ5 sϪ1) was efficiently accelerated 3.8 ϫ 104-fold (with an observed rate constant or kobs of 0.65 sϪ1) when 2 ␮M of the DH-PH domain of LARG was mixed in the stopped-flow apparatus with 0.1 ␮M RhoA-mantGDP and 20 ␮M GDP (Fig. 2B).

Results

Conclusion