The Man Rhogdi2, 7 Site-directed Mutagenesis Of The Activity Of The Body And Its Expression Pattern Analysis In Human Breast Cancer

RAS GTPases form a large superfamily. Together with their associated effectors, they participate in a variety of cellular processes ranging from growth control, cytoskeletal rearrangement, cell survival to intracellular trafficking regulation. They are also involved in many human diseases including cancers. The family has been tentatively classified into five subfamilies: Ras, Rho, Rab, Ran and Raf, based on structure, biochemical and functional similarities. They work as molecular switches by cycling between GTP-bound "on" state and GDP-bound "off" state. Several regulators tightly regulate the cycling between these two states. One class of these regulators, named RhoGDIs (Rho GDP-dissociation inhibitors), can form complex with these small GTPases and thereby keep them in an inactive station by preventing the exchange of GDP bound to these GTPases for GTP.Rho GTPases have key function in signaling pathways, such as JNK and Rho-ROCK. Therefore, it is important for them to keep balance between GTP-bound "on" state and GDP-bound "off" state. Over expression or activation of Rho GTPases always results in gene transcription, cell growth and invasion. In addition, GDP-bound Rho GTPases has found to form complex with RhoGDIs in quiescent cells. Thus, the inactive state of Rho GTPases contributes significant importance.As one of key regulators of Rho GTPases, RhoGDI2 also invole in cell growth. RhoGDI2 has been shown to be a tumor related gene in several cancers. In 2002, Gildea et al firstly reported that RhoGDI2 inhibits metastasis in bladder cancer cell line; the role of RhoGDI2 in cancer attracts a lot of interest since then. While some groups found that RhoGDI2 is a cancer metastasis inhibitor at least in bladder and colon cancers, other reports showed increased expression of RhoGDI2 in breast and ovarian cancers. In addition, a recent paper by Zhang et al demonstrated RhoGDI2 promotes cancer cell invasiveness. Although the relationship of RhoGDI2 and cancer is still controversial, more and more scientists are interested in this gene.Based on previous reports and crystal structure of RhoGDI2, we chose 7 conserved residues to study the structural basis of RhoGDI2. The mutants were constructed using site-directed mutagenesis and were expressed in E. Coli, followed by purification. We then tested the inhibition of GDP dissociation as well as the binding ability to RhoA. We found that of seven site-directed mutants we made, LL(52,53)SS shows decreased binding ability to RhoA. To our surprise, all of the mutants exihibit impaired inhibition of GDP dissociation from Rho. More strikely, SL(44, 45)AA, LL(52, 53)SS, DD(181, 182)AA almost lost their function. Taken together with the high conservation along RhoGDI family, this indicated that the residues play critical roles in RhoGDI2.We detected the expression level of RhoGDI2 in tissues from 71 breast cancer patients as well as 5 breast cancer cell lines with different ability of metastasis. We found that the RhoGDI2 exhibits biphasic expression pattern (increase first then decrease) in both cell lines and cancer patients, suggesting that RhoGDI2 is involved in both tumorgenesis and cancer metastasis. This result may also explain the controversial results in the relationship of RhoGDI and cancer.In addition, we compared mRNA expression level of several other RAS GTPases including RhoC, CDC42, Rac2 and Rac3 using semi-quantitive RT-PCR. Consistent with other reports, we found the expression RhoC is increased in breast cancer compared to normar tissue, whereas there is no significant change of Rac2 and CDC42. To our interest, cancer patients showed obvious increase of Rac3 (17/17). This data indicated that similar to RhoGDI2, Rac3 may act as a potential tumor marker and further experiments are needed to clarify the function of Rac3.Ras superfamily is comprised of many members, and most of them have been reported playing a role in tumorgenesis and/or cancer metastasis. We found an uncharacterized RAS-related gene, named RasL10B, while searching NCBI database. Bioinformatic analysis showed that this protein may play a role in cancer. To study the potential role of this gene, we isolated cDNA condigng sequence from human leukocyte cDNA library. The RasL10B was mapped to human chromosome 17q12. It encodes a 203 amino acid protein with a molecular mass of about 23.2 kDa. The coding sequence of RasL10B was cloned into pET30a vector, expressed in Rosette(DE3) and purified to a homogenicity by Ni-NTA affinity chromatography, which provides an example for future study. RT-PCR analysis showed that RasL10B is expressed extensively in 7 human tissues, especially high in brain, pancreas, thymus and prostate. Subcellular location analysis of GFP-RasL10B fusion protein revealed that RasL10B was distributed to the cytoplasm of COS7 cells. Finally, the mRNA levels of RasL10B were down-regulated in human breast tumor cell lines of MCF-7, MDA-MB-468,MDA-MB-231 and MDA-MB435, compared with human normal breast epithelia cell line HBL100. Thus, RasL10B is a new member of Ras superfamily with tumor suppressor potential.