Ras-proteins

Ras proteins and the catalytic domain ofGa have similar three-dimensional structures... 

Figure 13.5 A Mg + atom links GTP to the Ras protein. Mg Is coordinated to one oxygen atom each from the p and y phosphates of GTP as well as to the side chains of Set 17 and Thr 35 of Ras. Two water molecules complete the octahedral coordination of Mg.

The farnesylation and subsequent processing of the Ras protein. Following farnesylation by the FTase, the carboxy-terminal VLS peptide is removed by a prenyl protein-specific endoprotease (PPSEP) in the ER, and then a prenylprotein-specific methyltransferase (PPSMT) donates a methyl group from S-adenosylmethionine (SAM) to the carboxy-terminal S-farnesylated cysteine. Einally, palmitates are added to cysteine residues near the C-terminus of the protein. 

Ras is a G protein that cycle between two conformations, an activated Ras-GTP or inactivated form Ras-GDP. Ras, attached to the cell membrane by lipidation, is a key component in many signalling cascades, which couple growth factor receptors to downstream effectors that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis and cell migration. Mutations and dysregulations of the Ras protein leading to increased invasion and metastasis, and decreased apoptosis are very common in cancers. 

The kidney contains the major site of renin synthesis, the juxtaglomerular cells in the wall of the afferent arteriole. From these cells, renin is secreted not only into the circulation but also into the renal interstitium. Moreover, the enzyme is produced albeit in low amounts by proximal tubular cells. These cells also synthesize angiotensinogen and ACE. The RAS proteins interact in the renal interstitium and in the proximal tubular lumen to synthesize angiotensin II. In the proximal tubule, angiotensin II activates the sodium/hydrogen exchanger (NHE) that increases sodium reabsorption. Aldosterone elicits the same effect in the distal tubule by activating epithelial sodium channels (ENaC) and the sodium-potassium-ATPase. Thereby, it also induces water reabsotption and potassium secretion. 

Hancock JF (2003) Ras proteins different signals from different locations. Nat Rev Mol Cell Biol 4 373-384... 

Ras Mutations yielding hyperactive Ras protein are frequent in human cancers... 

In both worm and fly, the Ras protein acts as a switch that determine cell fate. In C. elegans, the activation of Ras determines the formation of vulval as opposed to hypodermal (skin) cells (for sequence of events, see Table 8.4). In Drosophila photoreceptors, the activation of Ras determines the development of R7 as a neuronal as opposed to a cone cell. In both cases, Ras proteins operate downstream of receptor tyrosine kinases that are activated by cell-cell interactions. 

The quantum yield of the mant fluorophore in mant-GDP or mant-GTP increases approximately by 100 % when the molecule changes from the aqueous environment into the nucleotide binding pocket of the Ras protein. Therefore the kinetics of complex formation between nucleotide free Ras and the mant analogues of GDP or GTP could be detected easily in a stopped flow system by an increase in fluorescence signal. 

Fig. 14A-C. Guanosine-nucleotides for biophysical characterizations of Ras proteins A gua-nosinediphosphate with a methylanthraniloylester (in grey) at the 3 -position of the ribose B guanosine-5 -[/3,y-imido]-triphosphate (GppNHp) C guanosine-5 -0-(3-thiotriphosphate)...

The hydrolysis of mant-GTP bound to Ras can be monitored by a slight decrease in fluorescence. Binding experiments of N-Ras with the non-hydro-lyzable GTP-analogue mant-GppNHp showed a biphasic increase in fluorescence. The slow phase had the same amplitude as the decrease observed for the hydrolysis of Ras mant-GTP which led to the hypothesis that a conformational change in the Ras protein proceeds GTP-hydrolysis [170] and represents the rate limiting step ... 

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