Abstract
Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.
Highlights
Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins
We have previously shown that FGN binding and degradation by CHO cells transfected with Mac-1 is inhibited by LPM19c, a monoclonal antibody (mAb) that blocks FGN binding to Mac-1, confirming the role of Mac-1 in this degradation pathway [17]
CHO cells transfected with p150,95, a known fibrinogen receptor [23], bound and degraded FGN, and this FGN turnover was inhibited by IB4, an anti-CD18 mAb that blocks CD11/CD18 function
Summary
Materials—Two-chain active urokinase and an amino-terminal fragment (residues 1–143, ATF) of urokinase were gifts of Dr Jack Henkin (Abbott Laboratories, Abbott Park, IL). To investigate the effect of uPAR occupancy on FGN binding and degradation, incubations with transfected CHO cells were performed in the presence of ATF (5 nM). For CHO cells transfected with Mac-1, p150,95, or chimeric versions of Mac-1 and p150,95, FGN turnover inhibitable by the anti-CD11b I-domain mAb LPM19c (Mac-1, MP-1–5) or anti-CD18 mAb IB4 (p150,95 and MP-6) was calculated and expressed relative to Mac-1/uPAR CHO cells in the absence of ATF. Passage 1–3 human saphenous vein smooth muscle cells (SMC) [105] were seeded into Transwell inserts (Becton Dickinson) precoated on the bottom with fibronectin (10 g/ml) or collagen type I (20 g/ml) and cultured overnight with or without peptide M25 (25–100 M) or scM25 (100 M). All assays were performed in triplicate, and the data were expressed as percentage of inhibition by the peptides
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