The EMBO Journal vol.14 no.22 pp.5550-5556, 1995 Critical amino acid residues for ligand binding are clustered in a predicted n-turn of the third N-terminal repeat in the integrin a4 and a5 subunits Atsushi Irie, Tetsuji Kamata, inflammation, inflammatory bowel disease) (for review, Wilma Puzon-McLaughlin and see Lobb and Hemler, 1994). Therefore, the ligand-a4,1l Yoshikazu Takada1 integrin interaction will be a therapeutic target for many diseases. Understanding of the ligand binding mechanism Department of Vascular Biology, The Scripps Research Institute, and identification of ligand binding sites are important for 10666 North Torrey Pines Rd, La Jolla, CA 92037, USA designing inhibitors that modulate the interactions. We 'Corresponding author recently localized the putative ligand binding site of the a4 subunit by mapping epitopes for function-blocking Integrin a401 is a receptor for vascular cell adhesion antibodies (Kamata et al., 1995). Anti-ax4 mAbs that block molecule (VCAM)-1 and fibronectin (CS-1). The a401- VCAM-1 and CS-I to a411 were mapped within residues ligand interaction is involved in the pathogenesis of 108-268 of ax4, suggesting that VCAM- I or CS-I binding diseases and is, therefore, a therapeutic target. Here, sites are close to or overlapping within x4. we identified critical residues of a4 for ligand binding In the present study, we have identified critical residues using alanine-scanning mutagenesis of the previously of ax4 for VCAM-1 or CS-I binding to cx4p1 within the localized putative ligand binding sites (residues 108- putative ligand binding site of x4 using alanine-scanning 268). Among 43 mutations tested, mutations of Tyrl87, mutagenesis (Cunningham and Wells, 1989). Mutations Trpl88 and Glyl90 significantly inhibited cell adhesion of Tyrl87, Trpl88 and Gly190 significantly reduced both to both VCAM-1 and CS-1. This inhibition was not VCAM-1 and CS-1 binding. These critical residues are due to any gross structural changes of a44p1. These clustered in a predicted 1-turn structure (residues 181- critical residues are clustered in a predicted ,3-turn 190) of the third N-terminal repeat of the ax4 subunit structure (residues 181-190) of the third N-terminal (Tuckwell et al., 1994). The repeat does not involve the repeat in a4. The repeat does not contain divalent putative cation binding motifs. We show that mutations cation binding motifs. Notably, the mutations within within the corresponding region of a5 significantly affect the corresponding region of aS significantly reduced fibronectin-aS,15 interaction, suggesting that the predicted fibronectin-a5jx1 interaction. These findings suggest 5-turn structure of the ax subunit may be ubiquitously that the predicted 13-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins. involved in ligand binding of non-I domain integrins. Keywords: fibronectin/integrin/ligand binding/mutagenesis/ VCAM-1 Results Mutations of Tyr187, Trp188 and Gly190 of a4 in a predicted f-turn affect a4/81-specific cell adhesion Introduction to VCAM-1 and CS-1 To identify amino acid residues on the W4 subunit critical Integrins are a superfamily of cell surface heterodimers, for ligand binding, we introduced multiple mutations which mediate signal transduction through interactions within the previously located putative ligand binding site with cellular or extracellular ligands (Yamada, 1991; Hogg of cx4 (residues 108-268) (Kamata et al., 1995). CHO et al., 1992; Hynes, 1992; Hemler et al., 1994; Springer, cells were co-transfected with either wild-type or mutant 1994). a41l integrin recognizes vascular cell adhesion human W4 cDNA in an expression vector, together with molecule (VCAM)- 1 (Elices et al., 1990), which is the expression vector containing the neomycin resistance expressed on activated endothelial cells and constitutively gene. Transfected cells were selected for G418 resistance. on bone marrow stromal cells (Osborn et al., 1989; Rice Typically 20-50% of the G418-resistant CHO cell popula- and Bevilacqua, 1989), and the alternatively spliced IIICS tions stably express human W4, as detected by flow portion [connecting segment-I (CS-1)] of fibronectin cytometry. Human ca4 is expressed as a heterodimer with (Wayner et al., 1989; Guan and Hynes, 1990; Mould endogenous hamster 131 (Kamata et al., 1995). et al., 1990, 1991). Evidence is accumulating that a4,1 Stably transfected cells were then tested for their ability plays a central role in leukocyte recruitment (for review, to adhere to different substrates. CHO cells stably see Lobb and Hemler, 1994). a41l integrin has been expressing human W4 were used without further enrich- shown to initiate lymphocyte contract ('tethering') in vitro ment for a4 expression. Untransfected CHO cells adhere with vascular endothelial cells under shear and in the to fibronectin well (>90 %) due to endogenous c5131 absence of a selectin contribution (Alon et al., 1995; Berlin (Takada et al. 1992), but do not adhere significantly to et al., 1995). Anti-a4 monoclonal antibodies (mAbs) have bovine serum albumin, VCAM-1 or CS-1. In contrast, shown therapeutic effects in numerous animal models cells expressing wild-type W4 adhere to both VCAM-1 of disease (e.g. experimental allergic encephalomyelitis, (Figure 1B) and CS-1 (Figure 1B). Most CHO cells contact hypersensitivity, non-obese diabetes, allergic lung expressing W4 mutants showed binding to either VCAM-1 555050 Oxford University Press Ligand binding to integrins ac4p1 and a5ft1 A c o 1.6 2 1.4 0. *x 1.2- 0 o 1- m 0.8- . 0.6- 'a 0 0.4- O 0.2- co-m X,Z ILa zeXaco o MutaIL °s°a ° N N N N N t 0a Mutations B I- U) Or-V V -v v -v V .v -v-v -EV -V -V -V,.v -C mc C4 CM Cd0 Cd4 cm CIA Y i"z E> ibdlZY 0aOO )10(3c MuaiOU.nIs a e > Z Z h > Y a 0 > > e m a a X Mutations Fig. 1. Effects of mutations of a4 on binding to VCAM-I (A) or CS-I (B). CHO cells expressing wild-type or mutant Ca4 were incubated for 1 h at 370C with VCAM-l-Cc fusion protein (A) or CS-1-RSA (B) immobilized on to plastic wells (at a coating concentration of 0.05 and 0.025 jig/well, respectively). After rinsing the wells to remove unbound cells, the adherent cells were quantified using an endogenous phosphatase assay. Data are expressed as means (% of bound cells per % of a4 positive cells) + SD of triplicate experiments. Transfected CHO cells after G418 selection were used without further enrichment of the a4-positive population, except that CHO cells expressing wild-type aX4 are clonal. Expression of a4 was determined by flow cytometry with mAb B5G10. More than 22% of the G418-resistant CHO cells were B5G1O positive. Some a4 mutants (F19A, R1OOA, Kl 16A, D138A, T199A, F227A and D281A) were not expressed on the surface of cells. or CS-1 at levels comparable with cells expressing wild- substrates (Figure 2A and B). The G 190A mutant [mean type a4. Interestingly, the Y187A and G190A a4 mutants fluorescent intensity (MFI) for a4 expression, 262] did showed significantly lower adhesion to both VCAM- 1 and not show significant adhesion to either ligand, even at CS- I than wild-type or the other mutant a4s. The adjacent high ligand coating concentrations (Figure 2). The Y187A W188A mutant a4 also showed slightly lower binding to mutant showed significantly lower adhesion to VCAM-1 both ligands. or CS-1 than did wild-type a4, especially at a low coating Interestingly, Tyrl87, Trpl88 and Gly190 are within concentration of substrates, although the expression level the predicted P-tum structure (residues 181-190 of a4) of the Y187A mutant (MFI for a4 expression, 516) is of the third N-terminal repeat (Tuckwell et al., 1994). higher than that of the wild-type (MFI for a4 expression, CHO cells expressing a4 with mutations at positions 185- 209). The Y187A mutant was significantly slower than 190 and 202 were cloned by cell sorting and further wild-type cx4p1 in establishing adhesion to VCAM-1 or characterized. The adhesion of the cloned CHO cells CS- I (data not shown). The W 188A mutant showed lower expressing the c4 mutants to VCAM- I or CS- 1 was adhesion to both ligands at low substrate concentrations determined as a function of coating concentrations of than did wild-type a4, but this mutation was much less 5551 A.Irie et al. A 80 mn60 8go6 c40 0 oA20 0 E z 0 0 0 0.02 0.04 0.06 0.08 0.1 VCAM-1 (,ug per well) 0 B cc 100 80 0 60 c 3 40 m 0AO 20 Fluorescent Intensity 0 Fig. 3. The Y187A, W188A and GI9OA mutations block binding of 0 0.02 0.04 0.06 0.08 0.1 soluble VCAM-l to a411. CHO cells were incubated with soluble CS-1 per VCAM-1-CK fusion protein (900 ng) in DMEM for 30 min on ice. (gg well) Bound VCAM-1 was detected with FITC-labeled goat anti-mouse CK Fig. 2. Adhesion of CHO cells homogeneously expressing o4 mutant IgG using flow cytometry. The S185A, S186A, T189A and Y202A mutants showed binding profiles similar to that of az4. to VCAM-1 (A) and CS-1 (B) as a function of coating concentration wild-type The binding of VCAM-1 to wild-type was blocked of substrates. Clonal CHO cells expressing wild-type (0) or mutant a4,B1 by 10 mM EDTA in the medium. The S185A, S186A and T189A mutant a4s showed a4 were incubated for 1 h at 37°C with VCAM-1-Cic fusion protein similar binding profiles to that of (A) or CS-1-RSA (B) immobilized at different concentrations (up to wild-type x4 (data not shown).