Mechanistic Insights into Specificity, Activity and Regulatory Elements of the RGS-containing Rho-specific Guanine Nucleotide Exchange Factors p115, PDZ-RhoGEF (PRG) and Leukemia- associated RhoGEF (LARG)

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, PRG and LARG. We demonstrate that (i) these GEFs are specific exchange factors for the Rho isoforms (RhoA, RhoB, RhoC) and inactive towards 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 10⁷-fold, which is at least as efficiently 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 (RGS) and the DH domains, revealed a significantly reduced GEF activity supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs.