The Function Of Rap1 And Ras In The Adherens Junctions

H-Ras and Rap1 belongs to the Ras superfamily small GTPase protein, regulating essential biological activities including cell proliferation, differentiation, apoptosis, cytoskeletal rearrangement. They were found to antagonize each other in several different biological functions recently. Both of them are localized at the cell membrane and cell junction sites, and regulate adherens junctions antagonistically. In order to approach the antagonistic mechanism of Rap1 and H-Ras during the formation of adherens junctions, the change in intracellular localization and interacting proteins of Rap1 and H-Ras in breast cancer cell lines MCF7 following calcium-switching reconstruction of adherens junctions were explored.Under normal culture conditions, GFP-H-ras, GFP-Rap1 and E-cadherin showed clear plasma membrane localization, and enriched in cell junction sites in MCF7 cell. Rap1 and E-cadherin were also found to be localized at cytosol along with the plasma membrane. When treating with low Ca2+ medium, localization of Rap1 and E-cadherin in plasma membrane decreased significantly, following the reduced pseudopodium, cell-cell contact. However the plasma membrane localization of GFP-H-Ras was not disturbed by adding low Ca2+ medium. When changing to normal culture conditions, Rap1 relocated to the membrane earlier than E-cadherin. Those result suggested that Rap1 may recruit E-cadherin to cell membrane, and positively regulate the formation of E-cadherin-dependent adherens junctions. And the H-Ras may not regulate directly adherens junctions through modulating the localization of E-cadherin.Subsequently, the interacting proteins of H-Ras and Rap1 before and after Ca2+ switching were isolated by pull down assay, and the binding ability of PI3K to H-Ras and Rap1 were estimated. The results indicated that binding of PI3K to H-Ras was decreased, following Ca2+ switching, while the binding to Rap1 was increased. Totally, our results suggested that H-Ras and Rap1 may regulate cell-cell adherens junctions formation through differentially modulating the E-cadherin localization and different binding ability to the downstream factors.