Activation of Rap1, Cdc42, and rac by nectin adhesion system.
Nectin is an immunoglobulin-like cell-cell adhesion molecule and forms adherens junctions cooperatively with cadherin. Trans-interaction of nectin induces activation of Rap1, Cdc42, and Rac small G proteins. The activity of these small G proteins can be analyzed by the pull-down assay using GST-Ral-GDS fusion protein for Rap1 and GST-PAK-CRIB for Cdc42 and Rac. The fluorescent resonance energy transfer (FRET) system is also available to spatially and temporally detect the activity of these small G proteins in the living cells. In addition to these assays, the activity of Cdc42 and Rac is indirectly, but easily, evaluated by the cell-spreading assay to examine formation of filopodia and lamellipodia, respectively. To clearly explore the effect of trans-interacting nectin on the small G proteins, we use L fibroblasts stably expressing nectin-1 (nectin-1-L cells) and the extracellular domain of nectin-3 fused to human IgG Fc (Nef-3). Treatment of nectin-1-L cells with Nef-3 remarkably increases both the amount of the GTP-bound form and the FRET efficiency of all Rap1, Cdc42, and Rac small G proteins. In the cell-spreading assay, Cdc42 and Rac activated in this way promote the formation of filopodia and lamellipodia, respectively. Here, we focus on how the activity of Cdc42 and Rac induced by trans-interacting nectin is examined by use of the pull-down and the cell-spreading assays.
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine, Japan.
Hisakazu
H
Ogita
Yoshimi
Y
Takai
D000818:Animals; D015815:Cell Adhesion Molecules / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D007739:L Cells; D051379:Mice; D000075983:Nectins; D016900:Neurofilament Proteins / Q000378:metabolism; D015398:Signal Transduction; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; C000619014:NECTIN3 protein, human; C000619015:Nectin1 protein, mouse; C000619017:Nectin3 protein, mouse; D000075983:Nectins; D016900:Neurofilament Proteins; C061195:neurofilament protein M; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins