Tress of two dynes/cm2. Cells treated with 5 mM EDTA have been applied as a control. Information are mean S.E. (n 3). p values have been calculated by one-way ANOVA with Dunnett post-tests. ***, p 0.001. C and D, differential interference contrast and interference reflection microscopy photos of CHO-K1 steady transfectants that adhered to immobilized MAdCAM-1 in 1 mM Ca2 / Mg2 (C) or 0.5 mM Mn2 (D). The pictures are representatives from one of 3 independent experiments. Scale bars 50 m. E, quantification of cell spreading region (projection on substrates) of 4 7 CHO-K1 transfectants around the basis of differential interference contrast images. Information are imply S.E. (n 50). p values were calculated by two-way ANOVA with Bonferroni post-tests. ***, p 0.001. F, CHO-K1 cells stably expressing WT or mutant four 7 were plated on poly-L-lysine in serum-free Ham’s F12 medium or on MAdCAM-1 in Ham’s F12 medium containing 1 mM Ca2 /Mg2 or 0.5 mM Mn2 for two h, lysed, and blotted for indicated molecules. Phosphorylated Y397-FAK and Y118-paxillin were blotted as indications of FAK and paxillin activation, respectively. A representative outcome of 3 independent experiments is shown.Yet another notable getting of our study is that the disulfide bond-stabilized W1 4- 1 loop in the 4 -propeller domain is essential for the global conformational rearrangement of 4 7 triggered by inside-out signaling. Studies have shown that the nearby conformational modifications inside the integrin head domain are closely related with its global conformational rearrangements (27, 31, 44 ?46). Mutations about the ligand binding site of integrin could affect the global conformational alterations of integrin molecules (27, 31, 44 ?46). In this study, as the W1 4- 1 loop is situated around the face on the -propeller domain, which binds the I domain and forms the ligand-binding web-site of integrin, it is tempting to speculate that deletion or mutations of this loop could exert some effects on the ligand-bindingMAY 17, 2013 ?VOLUME 288 ?NUMBERinterface formed between the subunit -propeller domain plus the I domain, which may possibly impact the regional conformational changes within the head domain of integrin that happen to be required by the worldwide conformational rearrangements triggered by inside-out signaling.439579-12-1 web This study also finds that the disulfide bond-stabilized W1 4- 1 loop is expected for four 7-mediated cell spreading and outside-in cell signaling (Fig.2-Bromo-3-fluoropyrazine Data Sheet 6).PMID:23805407 Integrin-mediated cell spreading is a complex procedure that entails diverse signaling networks (47). Among the early actions in transducing extracellular cues via integrins for the cytoskeleton could be the activation of FAK and paxillin (47). Our study showed that the W1 4- 1 loop mutations decreased the activation of each FAK and paxJOURNAL OF BIOLOGICAL CHEMISTRYW1 4- 1 Loop RegulatesFunction7. Gorfu, G., Rivera-Nieves, J., and Ley, K. (2009) Role of 7 integrins in intestinal lymphocyte homing and retention. Curr. Mol. Med. 9, 836 ?850 eight. Hamann, A., Andrew, D. P., Jablonski-Westrich, D., Holzmann, B., and Butcher, E. C. (1994) Function of 4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152, 3282?293 9. Lanzarotto, F., Carpani, M., Chaudhary, R., and Ghosh, S. (2006) Novel treatment possibilities for inflammatory bowel disease. Targeting 4 integrin. Drugs 66, 1179 ?189 10. Chen, J., Salas, A., and Springer, T. A. (2003) Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster. Nat. Struct. Biol. 10, 995?001 11. Chen, J., Takagi, J., Xie, C., Xiao, T., Luo, B.