GTP-binding protein, Ras

Figure 3. MAP kinase regulatory pathway. The MAP kinase signaling pathway begins with activation of the receptor tyrosine kinase (RTK) by exogenous signals, such as growth factors and insulin. The signal is then transmitted into the cell via activation of the Raf serine/threonine kinase either directly by the RTK or through the GTP-binding protein, Ras. The signal is then transmitted to the nucleus and to other cytoplasmic proteins via MAPKK and MAPK.

More recently it has become clear that also the function of G proteins can be altered. There are a small number of proteins that have been found to associate with G proteins and to affect their function. These proteins include the growth cone associated protein GAP-43, which has been found to enhance GTP binding by Gq in a manner similar to receptors [26], and a complex between the small GTP-binding protein ras p21 and its GTPase activating protein (ras-GAP) which impair coupling of muscarinic receptors to potassium channels [27]. 

Recent studies of chemotaxis signaling in Dictyostelium, which shares many features with the mammalian chemotaxis signaling pathway (Chung et al, 2002 Devreotes et al, 2003 Merlot et al, 2003) suggest that activation of PlSKy may involve not only the Gj3y complex (Lopez-Ilasaca et al, 1997) but also the small GTP binding protein Ras (Fig. 3) (Chung et al, 2002). Both the mammalian and PI 3K isoforms utilized in chemotaxis... 

For instance, if the hormone adrenalin arrives at a liver cell and binds to the adrenalin-specific receptor embedded in its membrane, this leads to a whole cascade of reactions involving a G-protein, an effector catalysing the synthesis of cyclic adenosin monophosphate (AMP) and eventually results in the export of glucose from the cell (Fig. 2.4). Another GTP binding protein, Ras, is important for the regulation of cell growth and differentiation. (Mutations of this protein are notorious for causing a variety of cancers.)... 

Five of the six loop regions (G1-G5 in Figure 13.4) that are present at the carboxy end of the p sheet in the Ras structure participate in the GTP binding site. Three of these loops, G1 (residues 10-17), G3 (57-60), and G4 (116-119), contain regions of amino acid sequence conserved among small GTP-binding proteins and the Ga subunits of trimerlc G proteins. 

The discovery of PLCs reveals a fourth mechanism whereby the enzyme can be activated (Fig. 20-5). PLCs possesses two Ras-binding (RA) domains in its carboxyl terminal region, and occupancy of these by Ras-GTP results in activation of the enzyme. In addition, the enzyme possesses a CDC-25 domain at its N-terminus, which serves as a guanine nucleotide exchange factor (GEF) for small GTP-binding proteins such as Ras or RaplA. Thus PLCe can not only activate the GDP-bound forms of these small GTP-binding proteins but can also be... 

All GTP binding proteins in signal transduction share a common structural element - the Ras-like domain which is responsible for the specific complexation of guanosine diphosphate and -triphosphate and which contains catalytic residues that promote GTP-hydrolysis. 

The Ras superfamily of GTP-binding proteins is composed of several subfamilies [7] which all contain the Ras-like domain of approximately 160 amino acids and 5 consensus sequences. Two of these highly conserved motifs are responsible for specific recognition of the guanosine nucleotide, and three are necessary for binding of the phosphate groups and complexation of a Mg++ ion, which is found in all Ras-like proteins. 

Ran (the Ras-related nuclear protein) is the major regulator of nucleo-cytoplas-mic transport [134] across the nuclear pore complex (NPC). Like other small Ras-like GTP-binding proteins, it switches between a GTP- and a GDP-bound form by GTP-hydrolysis and nucleotide exchange [135]. In contrast to its relatives, Ran does not undergo posttranslational modification. 

Ras and its relatives are subjects of intensive investigations by biological, biochemical, biophysical, and medical studies. Within just one decade more than 17,000 articles (Medline, 1966-2000) deal with function and properties of this protein. Structural and functional data, based on Ras as a prototype, have provided insight into the basic principles of GTP-binding proteins, their activation, de-activation, and signal transmission. 

Inhibition of the lipid modification cascade provides an alternative way to block aberrant signaling pathways and that opportunity can be exploited in anticancer therapy. As part of the growth factor, signaling of the false activation is transmitted by the mutated ras gene encoded proteins (Ras) and ultimately leads to uncontrolled cell growth. These typical GTP binding proteins are also subject to membrane anchoring and the biosynthesis of those Ras proteins can be blocked at the posttranslational prenylation step. 

Eukaryotic cells utilize an efficient transport system that delivers macromolecules fast and secure to their destination. In the case of the small GTP binding proteins of the Ras family the modified C-terminus seems to be sufficient for addressing the polypeptide to its target membrane (in the case of Ras itself the plasma membrane). Lipopeptides with the C-terminal structure of N-Ras (either a pen-tamer with a C-terminal carboxymethylation and farnesylation or a heptapeptide with a palmitoyl thioester in addition) and a N-terminal 7-nitrobenz-2-oxa-l,3-diazolyl (NBD) fluorophore were microin-jected into NIH3T3 fibroblast cells and the distribution of the fluorophore was monitored by confocal laser fluorescence microscopy. Enrichment of the protein in the plasma membrane was efficient only for peptides with two hydrophobic modification sites, while the farnesylated but not palmitoylated peptide was distributed in the cytosol.1121... 

By combining bacterial expression and chemical synthesis Ras constructs with the properties of the post-translationally modified protein can be generated. These hybrid proteins can insert into artifical and biological membranes, have been proven to be efficient tools for biochemical, biophysical and biological experiments and can be synthesized in large amounts. Principally the same method is applicable to many of the Ras-related GTP-binding proteins or the y-subunit of heterotrimeric G proteins. 

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