Structure of the Human ras Gene Family
To study the structure of the ras gene family, we devised an original oligonucleotide strategy and isolated cDNAs for several new ras-related proteins: the ra1A protein (50% a.a. identity with ras) and the rab proteins, related to the yeast YPT and SEC4 proteins. These new isolates, as well as drosophila D-ras3 and rho probes were then used to precise the structure of the ras family, in human.
At present, the ras family includes three main branches represented by ras, rho and rab genes. 1) The first branch includes rasH, rasK and rasN/ R-ras/ra1A and ra1B/rap1A and rap1B/rap2. 2) So far, the second branch only includes the rhoA, rhoB and rhoC proteins sharing 80–90% aa identity, it is likely that other proteins remain to be discovered in this branch. 3) The third branch includes rab1 (homologous to yeast YPT)/rab2/rab3A and rab3B/rab4/rab5/rab6 (homologous to the YPT2 protein of fission yeast). A large array of evidence coming from yeast studies implicate these proteins in secretion.
All ras-related proteins share four regions of high homology corresponding to the GTP binding site in positions 10–17, 57–63, 113–120 and 143–149 ; however significant differences are found in these regions: rho proteins have Gly 12 but not Gly 13, rab proteins have Gly 13 but not Gly 12 (rab3 and rab5 have neither Gly 12 nor Gly 13) and rap proteins have a Thr instead of Gln 61, suggesting that these proteins might differ in their GTPase activities and/or GTP/GDP exchange rates. All ras-related proteins possess a Cystein near the C-terminus but closely surrounding sequences are specific, suggesting a role in the different intracellular locations of these proteins. Other external regions are differentially conserved in each branch such as region 32–42, known as the putative “effector” region, for the ras branch (striclty identical in ras and rap 1 proteins) while the most conserved external loop in the rab proteins is around position 63–73.
We have expressed several of these proteins in E. Coli and confirmed their GTP binding ability; we are now studying their biochemical and functional specificities.
KeywordsGTPase Activate Protein Oligonucleotide Strategy
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