Mechanistic insights into specificity, activity and regulation of Rho exchange factors

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 role of this and other domains in the DH‐mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is not well understood. This 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, B, C) and inactive towards other member 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 107‐fold (iii) an N‐terminal region 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 a proposed model of the RGS‐mediated intramolecular autoinhibitory mechanism for p115‐like RhoGEFs. This work was supported in part by the DFG and Forschungskommission der Medizinischen.