Figure 2.

Ras is a GDP/GTP-regulated binary switch. (A) The three RAS genes encode four 188–189 amino acid proteins that share 82–90% overall sequence identity; KRAS encodes two splice variants due to alternative exon 4 utilization, leading to divergent C-terminal sequences. Exons 4A and 4B encode 39 and 38 amino acids, respectively, with 19 identical and 4 conserved substitutions. K-Ras4A is most similar to viral K-ras while K-Ras4B is the predominant isoform expressed in human cells. Residues 1–164 comprise the G domain that contains six conserved sequence motifs shared with other Ras superfamily and GTP-binding proteins. These motifs are involved in binding either phosphate/Mg²⁺ (PM) or the guanine base (G) of GDP and GTP. Residues in Switch I (aa 30–38) and II (aa 60–76) change in conformation during GDP/GTP cycling. The core effector binding domain (E; residues 32–40) and flanking sequences are involved in effector binding specificity. (B) Regulators of the Ras GDP/GTP cycle. RasGEFs stimulate GDP/GTP exchange. With the 10-fold higher cellular concentrations of GTP over GDP, the net result of RasGEF stimulation is formation of active Ras-GTP. Ras-GTP binds preferentially to downstream effectors. RasGAPs accelerate the intrinsic GTP hydrolysis activity of Ras to promote formation of inactive Ras-GDP. Shown are “classic” missense mutants of Ras proteins that have been useful for dissection of Ras function. The Ras(S17N) dominant negative sequesters and blocks RasGEF activity, preventing Ras activation. The G12V and Q61L mutations, found in human cancers, impair GAP-stimulated GTP hydrolysis. The T35S, E37G and Y40C effector domain mutants (EDMs) differentially impair effector binding. The T35S mutant retains efficient binding to Raf but not PI3K or RalGEF, whereas the E37G mutant retains efficient binding to RalGEF but not Raf or PI3K, and the Y40C mutant retains efficient binding to PI3K but not Raf or RalGEF.