Low Molecular Weight GTP-Binding Proteins

The feet that mutant Ras genes have been associated with a wide variety of human tumors suggests that they are responsible for the uncontrolled growth of tumor cells. Studies involving in vitro cell culture and transgenic animals have demonstrated the ability of 

Finally, Ras proteins are also involved in the control of normal growth and are activated in response to diverse extracellular stimuli that influence cell growth and differentiation. Such fectors include those that stimulate the growth of fibroblasts, e.g. EGF and PDGF, or hematopoietic cells e.g. BL-2, IL-3 and GM-CSF. The cell surfece receptors for these extracellular stimuli are typically receptor tyrosine kinases (RTKs) such as the EGF receptor or receptors diat have associated nomeceptor tyrosine kinases (Herrmarm, Nassar, 1996 Khosravi-Far, Der, 1994). 

The transformation of rodent fibroblasts by a variety of tyrosine kinase oncogenes also causes chronic elevation of Ras-GTP levels. It thus seems obvious that Ras proteins ate essential components of tyrosine kinase mediated rtritogenic signaling pathways. Ras proteins are activated in response to a wide variety of extracellular stimuli that activate receptor and receptor-associated tyrosine kinases TKs). 

The Ras proteins encoded by all three human ras genes are homologous in their first 164 amino acids but their last 25 residues are divergent, except for a cystein (residue 186) four amino acids fiom the C-terminus. Four GTP binding domains can clearly be defined in the stracture of the Ras proteirrs. 

Several site directed mutation studies have indicated that residues 26 to 45, which are conserved in all Ras proteins, are likely to specify an interacting surfece for (an) effector molecule(s), since neither guanine nucleotide nor membrane association was affected by these mutations while transforming activity was abolished. 

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Although none of the G-protein subunits contains regions that might obviously associate with a lipid bilayer, heterotrimeric G-proteins are associated with the cytoplasmic surface of the plasma membrane as is Ras p21 and some of the other low molecular weight GTP-binding proteins. This is apparendy due to the fact that the y-subunits of the heterotrimeric G-proteins are prenylated, as is ras, and at least some of the G-protein a-subunits, e.g. Gj subfamily. 

Aktories K (1990) Clostridial ADP-ribosyltransferases — modification of low molecular weight GTP-binding proteins and of actin by clostridial toxins. In Med. Microbiol. Immunol. 179 123-36... 

DTT (100 mM final concentration in steps 3 and 4) enhances the GTP binding ability of low-molecular-weight GTP-binding proteins. This improves considerably the signal-to-noise ratio as well as the sensitivity of the procedure (Gromov and Celis, 1994). 

Using the protocol described here, it is possible to detect several low-molecular-weight GTP-binding proteins. A representative P autoradiograph of low-molecular-weight GTP-binding proteins detected in an lEF 2D gel blot of total human keratinocyte proteins is shown in Fig. 1. 

Doucet, J.-P., and Tuana, B. S. (1991) Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle. J. Biol. Chem. 266, 17613-17620. 

Almost all receptor-mediated neutrophil functions are mediated via GTP-binding proteins (G-proteins), which provide the link between occupancy of plasma membrane receptors and the activation of intracellular enzymes, such as phospholipases and protein kinases. There are two groups of G-proteins those that are heterotrimeric and those with low molecular weight. 

In addition to the large, heterotrimeric G-proteins that have been shown to play a role in signal transduction, a superfamily of low-molecular-weight (LMW) GTP-binding proteins, which are structurally related to ras, has been shown also to play important roles in the regulation of cell function. These proteins were initially identified by molecular cloning techniques and... 

Figure 6.4. Mode of action of low-molecular-weight G-proteins. The raslike proteins normally bind GDP, but this may be exchanged for GTP via a process that may be assisted by guanine nucleotide exchange protein (GNEP). The GTP-bound ras protein may then interact with and activate its target protein (X). The activity of GTPase activating protein (GAP) may then assist to hydrolyse GTP to GDP, to inhibit ras activity.

Receptors for ADP, epinephrine, thromboxane, thrombin and PAF have been well characterized (27, 31, reviewed in other chapters). Membrane spanning receptors of epinephrine, t mbin and thromboxane are coupled to the ubiquitous GTP-hinding proteins. Platelets contain monomeric, low molecular weight G proteins as well as heterotrimeric membrane associated G roteins. GTP binding to e a-subunit of G-proteins fiicilitates die interaction with effector enzymes, resulting in the hydrolysis of GTP to GDP, which terminates its stimulatory role (29). 

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