Effects Of Rap1GAP On The Formation Of Adherent Junction

Objective: To reveal the role of Rap1GAP in the formation of adherent junction. Rap1GAP is the GTPase activating protein of Rap1, which is the monomeric GTP binding G-protein, Rap1GAP can greatly promote the GTPase activity of Rap1 and accelerate the hydrolysis of Rap1-GTP. Series of research results demonstrated that GTP-binding Rap1 can promote the formation of AJ. Some researcher found Rap1GAP could inhibit the formation of AJ, even destroy the junction of epithelial cells which had already formed. But,other researchers demonstrated Rap1GAP could not inhibit the formation of AJ but only slow down the process of AJ formation . So, more works are needed to illustrate the role of Rap1GAP in the formation of AJ, which have close relations with EMT (Epithelial mesenchymal transition) and the metastasis of cancer. To reveal the role of Rap1GAP in the formation of adherent junctions will further the understanding of the mechanism in metastasis of cancer and provide a new target for cancer therapy.Methods: 1) 293 and Hela cells were transfected respectively with GFP-tagged Rap1GAP or empty vector by lipofactamine reagent, stably expressing cells were screened with G418 and were further identified by fluorescent microscope and westernblot techniques. 2) Growth curves of Rap1GAP stably expressing group were compared with GFP stably expressing group and untransfected group. 3) The junctions between Rap1GAP expressing cells were examined by phase contrast microscope and immnofluorescent technique. 4) The location of Rap1GAP were examined, when Rap1GAP was cotransfeted with Gαz to 293 cells transiently. and the location of Rap1GAP were examined in 293-Rap1GAP cells during thecalcium chelation experiment. 5) The location of Rap1GAP and N-cadherin in 293-Rap1GAP cells were examined by immnofluorescent technique during the calcium chelation experiment. 6) The location of Integrin-beta1 in 293-Rap1GAP cells was examined by immnofluorescent technique during the calcium chelation experiment.Results: 1) 293 cells stably expressing Rap1GAP(293-Rap1GAP),293 cells stably expressing GFP(293-GFP),Hela cells stably expressing Rap1GAP ( Hela-Rap1GAP ) and Hela cells stably expressing GFP(Hela-GFP) were generated. 2) The growth curve results showed the proliferation of Hela-Rap1GAP was slow, compared with Hela-GFP and Hela cells. The proliferation of 293-Rap1GAP was not significant different from 293-GFP and 293 cells. 3) 293-Rap1GAP grew as a integral flat layer and N-cadherin accumulated to cell-cell junctions in 293 cells. 4) Cotransfected with active type of Gαz, Rap1GAP accumulated to the junctions between 293 cells. 30 min after the adding EGTA, Rap1GAP accumulate to the junctions between 293 cells. 5) Results of calcium chelation experiment and immunofuoresence experiment. (a) AJ of 293-Rap1GAP is destroyed at the time of 60 min after the addition of EGTA , On the contrast, the AJs of 293-GFP were destroyed at the time of 15 min after the addition of EGTA. (b) AJ reformed 30 min after the restoration of calcium, there were no significant difference between 293-Rap1GAP and 293-GFP. (c) Before the addition of EGTA, Rap1GAP localized predominantly in the cytosol; 15,30,45min after the addition of EGTA, Rap1GAP translocated from the cytosol to the juctions between 293 cells partly; 60 min after the addition of EGTA, Rap1GAP localized predominantly in the cytosol; 15min after the resoration of calcium, some of Rap1GAP translocated to the juctions between 293 cells; 15min after the resoration of calcium , Rap1GAP localized predominantly in the cytosol again. 6) Integrin-beta1 located at junctions between 293 cells and could not be destroyed by EGTA.Conclution: Our results demonstrated 1) 293 and Hela cell strains stably expressing GFP or GFP-Rap1GAP were achived. 2) Rap1GAP can inhibit the proliferation of Hela cells but not 293 cells. 3) Rap1GAP could not destroy the adherent junctions between 293 cells. 4) Rap1GAP canaccumulate to the junctions between 293 cells. 5) Rap1GAP accumulated to the junctions between 293 cells could inhibit the destruction of AJ by EGTA. 6) Integrin-β-1 was the component of tight junction.