Small GTPases exist in two states, depending on whether they are bound to GTP or to GDP. The GDP-bound form is inactive but is converted into the active form by exchange of the GDP for GTP. This reaction is mediated by proteins known as guanine-nucleotide exchange factors, or GEFs, which cause the GTPase to release GDP and to bind the more abundant GTP (Fig. 7.4). Sos, which is recruited to signaling pathways by the adaptor Grb2 (see Section 7-2), is one of the GEFs for Ras. The binding of GTP induces a conformational change in the small GTPase that enables it to bind to and induce effector activity in a variety of target proteins. Thus, GTP binding functions as an on/off switch for small GTPases.
This GTP-bound form does not remain permanently active but is eventually converted into the inactive GDP-bound form by the intrinsic GTPase activity in the G protein, which removes a phosphate group from the bound GTP. Regulatory cofactors known as GTPase-activating proteins (GAPs) accelerate the conversion of GTP to GDP, thus rapidly downregulating the activity of the small GTPase. Because of GAP activity, small GTPases are usually present in the inactive GDP-bound state and are activated only transiently in response to a signal from an activated receptor. RAS is frequently mutated in cancer cells, and the mutated Ras protein is thought to be a significant contributor to the cancerous state. The importance of GAPs in signaling regulation is indicated by the fact that some mutations in Ras found in cancer act by preventing GAP from enhancing the intrinsic GTPase activity of Ras, thus prolonging the duration for which Ras exists in the active GTP-bound state.
GEFs are the key to G-protein activation and are recruited to the site of receptor activation at the cell membrane by binding to adaptor proteins or to lipid metabolites produced by receptor activation. Once recruited, they are able to activate Ras or other small G proteins, which are localized to the inner surface of the plasma membrane via fatty acids that are attached to the G protein post-translationally. Thus, G proteins act as molecular switches, becoming switched on when a cell-surface receptor is activated and then being switched off. Each G protein has its own specific GEFs and GAPs, which help to confer specificity on the pathway.
7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms.
We have seen how receptors can recruit intracellular signaling proteins to the plasma membrane through tyrosine phosphorylation of the receptor itself or of an associated scaffold, followed by recruitment of SH2-domain-containing signaling proteins or adaptors, such as Grb2 (Fig. 7.5). A second mechanism for membrane recruitment of signaling proteins is via binding to small GTPases, such as Ras, following their activation. As described in Section 7-3, small GTPases are constitutively bound to the cytoplasmic surface of the plasma membrane due to their fatty acid modifications. Once activated by exchange of GDP for GTP, the activated GTPases bind to signaling proteins such as Sos, relocalizing the bound proteins to the plasma membrane (see Fig. 7.5).