Fig. 2. Mutation of homologous polar residues within the extracellular domain of ß4 prevents attachment of 6ß4 to laminin-5 and tyrosine phosphorylation after EGF treatment but not recruitment of HD components. (A) Amino acid substitutions to generate an attachment defective ß4 subunit, ß4(AD). Homology analysis was carried out with the extracellular domain of the ß3 integrin subunit. Asterisks indicate residues essential for ligand binding in integrin IIbß3 (Baker et al., 1997). Arrows represent sites of point mutation and alanine substitution. (B) Distribution of HD components in ß4(AD) cells. Cells were cultured for 24 hours on glass coverslips fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. Immunofluorescence microscopy was performed as described for ß4(-) and ß4(+) cells (see Fig. 1A). Mouse and rabbit antibodies are colored red while rat antibodies are colored green, colocalization is therefore represented by a yellow color. Narrow images under each figure represent z-sections of the image above (nuclei are stained blue with Hoescht dye) Scale bar: 10 µm. (C) Attachment of keratinocytes to laminin-5 in the presence of inhibitory antibodies to 3 integrin (P1B5) and/or cells in suspension were applied to 96-well plates coated with 10 µg/ml laminin-5 and incubated for 60 minutes at 37°C before unattached cells were washed off. Inhibitory antibodies and control IgG were supplemented at 10 µg/ml. Adherent cells were fixed, stained with 0.1% crystal violet and solubilized with 10% acetic acid. Cell number was quantified by measuring optical density at 570 nM (n=4). The bar chart shows adherence of ß4(+) cells (light shading) vs. ß4(AD) cells (dark shading). (D) Attachment of keratinocytes to laminin-5 at 4°C in the presence of ß4 inhibitory antibody (ASC-8). Cells in suspension were applied to 96-well plates coated with 10 µg/ml laminin-5 and incubated for 60 minutes at 4°C before washing off unattached cells. Inhibitory antibodies and control IgG were supplemented at 10 µg/ml. Adherent cells were fixed, stained with 0.1% crystal violet and solubilized with 10% acetic acid. Cell number was quantified by measuring optical density at 570 nM (n=4). The bar chart shows adherence of ß4(+) cells (light shading) compared with ß4(AD) cells (dark shading). (E) Tyrosine phosphorylation of the ß4 subunit after stimulation of cells with EGF. ß4(+) and ß4(AD) keratinocytes were growth factor starved for 16 hours then stimulated with 100 ng/ml EGF. At time intervals indicated (in minutes), cells were lysed and immunoprecipitated with ß4 mAb, 3E1. Western blots show tyrosine phosphorylation (mAb 4G10, upper panels) and total ß4 in immunoprecipitates (with rabbit polyclonal antiserum 1922, lower panels). (F) Laminin-5 secretion and processing by transduced cells. Cells were grown on plastic culture dishes with or without 2 ng/ml EGF. After 24 hours, cells were removed with 20 mM ammonium hydroxide and matrix was extracted with 8 M urea buffer before western blotting with a polyclonal laminin-5 antibody. Symbols to the right of the blot indicate the separate laminin-5 subunits with a (p) indicating a processed subunit. (G) Activation of p44/42 MAP kinase by EGF. Cells were growth factor starved and treated for 5' with 2 ng/ml EGF before lysis and western blotting with phospho-p44/42 MAP kinase antibody (upper panel). Blots were then stripped and reblotted with total p44/42 MAP kinase antibody (lower panel).