Oops around the face of -propeller domain that bind the I domain and contribute for the formation with the 4 7 ligand-binding pocket (28) (Fig. 1A). A particular a single will be the W1 4- 1 loop, that is stabilized by a disulfide bond that exists exclusively in 4/ 9 subfamily (28) (Fig. 1). Thinking about the structure specificity of this loop, we hypothesize that this disulfide bond-stabilized W1 4- 1 loop may well contribute to unique two-phase cell adhesion mediated by four 7. Here we demonstrated that the disulfide bond-stabilized W1 4- 1 loop is essential for rolling cell adhesion mediated by the low-affinity interaction in between inactive four 7 and MAdCAM-1 but not for firm cell adhesion supported by the high-affinity interaction among Mn2 -activated four 7 and MAdCAM-1. Either breaking the disulfide bond or deleting the disulfide bond-occluded segment within the W1 4- 1 loop not merely blocked the global conformational rearrangement and activation of four 7 triggered by talin or phorbol-12-myristate-13-acetate (PMA) by way of insideout signaling but also disrupted integrin outside-in signaling. Thus, the disulfide bond-stabilized W1 4- 1 loop is often a novel regulatory element of integrin affinity and bidirectional signaling and plays an important role in supporting the 4 7-mediated rolling adhesion.FunctionEXPERIMENTAL PROCEDURES cDNA Building and Expression–The 4 site-directed mutations were generated using QuikChange (Stratagene). WT human four cDNA in vector pcDNA3.1/Hygro(-) (Invitrogen) was utilized because the template. cDNA of the human talin head domain (talin 1?435) was cloned into vector pmCherry-C1 (modified from vector pEGFP-C1) to produce a construct in the talin head domain with N-terminal fused mCherry. All constructs had been confirmed by DNA sequencing. Transient Transfection of 293T cells was performed as described (10). CHO-K1 cells stably expressing WT and mutantMAY 17, 2013 ?VOLUME 288 ?NUMBERhuman four 7 were established by cotransfection of human 4/ 7 cDNAs and selection by 0.2 mg/ml hygromycin (Amresco). Antibodies and Reagents–The human integrin four 7-specific blocking mAb Act-1 was as described previously (29, 30). The rat mAb FIB504 against human 7 was ready applying a hybridoma (Developmental Research Hybridoma Bank, University of Iowa). Phycoerythrin (PE)-conjugated FIB504 and mAb to paxillin were from BD Biosciences.Tetrakis(triphenylphosphine)palladium Chemical name mAb to pY118-paxillin was from Cell Signaling Technologies, Inc.8-Bromo-1,6-naphthyridine uses , and mAbs to FAK and pY397-FAK were from Upstate Biotechnology, Inc.PMID:23319057 Alexa Fluor 488-conjugated goat anti-rat IgG mAb was from Invitrogen. Human MAdCAM-1/Fc fusion protein (h-MAdCAM-1/ Fc) was prepared as described previously (ten). Flow Cytometry–Immunofluorescence flow cytometry was done as described (31). The expression level of integrin 4 7 on transient 293T transfectants was determined by staining with mAb FIB504 and, subsequently, staining with Alexa Fluor 488conjugated mAb goat anti-rat IgG. The expression degree of integrin 4 7 on stable CHO-K1 transfectants was determined by staining with PE-conjugated FIB504. Stained cells have been then measured utilizing FACSCalibur (BD Biosciences) and analyzed using WinMDI software program. Flow Chamber Assay–The flow chamber assay was performed as described (10, 11). A polystyrene Petri dish was coated with a 5-mm-diameter, 20- l spot of 10 g/ml purified h-MAdCAM-1/Fc in coating buffer (PBS, ten mM NaHCO3 (pH 9.0)) for 1 h at 37 , followed by 2 BSA in coating buffer for 1 h at 37 to block nonspecific binding sites. Cells were washed twice with HBS (20 m.
