Brefeldin A

Abstract

The guanine nucleotide exchange factors (GEFs) for small G proteins first recognize the GDP-bound protein and lower the affinity for GDP, leading to a nucleotide-free transition state. GTP then enters the empty guanine-binding site and induces a conformational change that displaces the GEF (see Figure 7 of Peyroche et al. 1999 Peyroche A Antonny B Robineau S Acker J Cherfils J Jackson C.L Mol. Cell. 1999; 3 : 275-285 Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar ). For Ras, and probably for most other G proteins, this last step is rate limiting for the exchange reaction and the release of Ras-GTP ( Lenzen et al. 1998 Lenzen C Cool R.H Prinz H Kuhlmann J Wittinghofer A Biochemistry. 1998; 37 : 7420-7430 Crossref PubMed Scopus (185) Google Scholar ). The structure of the nucleotide-free form of Ras in complex with the Sos RasGEF domain has been solved, providing important insights on the details of these three steps ( Boriack-Sjodin et al. 1998 Boriack-Sjodin P.A Margarit S.M Bar-Sagi D Kuriyan J Nature. 1998; 394 : 337-343 Crossref PubMed Scopus (541) Google Scholar ). Extensive use has been made of mutant forms of Ras where serine 17 is replaced by asparagine (RasN17); the corresponding mutations in related G proteins (Rac, Rho, etc.) have also been widely used to dissect signal transduction pathways. These mutant proteins act as dominant negatives, blocking activation of endogenous Ras proteins by upstream activators. In the RasN17 mutant, the affinity for GTP is decreased by three orders of magnitude compared to wild-type Ras; thus, GTP is no longer able to bind and displace the GEF, resulting in an abortive complex between nucleotide-free RasN17 and the GEF. Because exchange factors are usually expressed at lower levels than their target G proteins, low levels of RasN17 proteins are sufficient to sequester all exchange factors into abortive complexes, preventing endogenous Ras activation.