Abstract
Circulating leukocytes are nonadherent but bind tightly to endothelial cells following activation. The increased avidity of leukocyte integrins for endothelial ligands following activation is regulated, in part, by interaction of the beta2 subunit cytoplasmic tail with the actin cytoskeleton. We propose a mechanism to explain how tethering of the actin cytoskeleton to leukocyte integrins is regulated. In resting leukocytes, beta2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the beta2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein alpha-actinin. The association of alpha-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. Glutathione S-transferase-beta2 tail fusion protein/mutagenesis experiments suggest that the affinity of alpha-actinin binding to the beta2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic alpha-actinin binding domain. Positive and inhibitory domains within the beta2 tail regulate alpha-actinin binding: a single 11-amino acid region (residues 736-746) is necessary and sufficient for alpha-actinin binding, and a regulatory domain between residues 748-762 inhibits constitutive association of the beta2 tail with alpha-actinin.
Highlights
Circulating leukocytes are nonadherent but bind tightly to endothelial cells following activation
Glutathione S-transferase-2 tail fusion protein/mutagenesis experiments suggest that the affinity of ␣-actinin binding to the 2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic ␣-actinin binding domain
Talin Constitutively Associates with the 2 Subunit in Unactivated PMNs—We previously reported that the protein ␣-actinin became associated with the cytoplasmic domain of the 2 subunit in PMNs following activation but that ␣-actinin did not interact with 2 integrins in unactivated cells (6)
Summary
PMN Isolation, Activation, and Extraction—Human PMNs were isolated from fresh citrate phosphate dextrose-anticoagulated blood obtained from volunteer donors using 6% dextran to sediment erythrocytes, followed by separation from lymphocytes by centrifugation in Histopaque 1077 (Sigma). PMN extracts for immunoblot analysis with anti-talin antibody 8d4 (Sigma) and for affinity binding studies were prepared by treating 107 cells with 1 ml of lysis buffer consisting of Tris-buffered saline (TBS) (50 mM Tris, pH 7.4, 150 mM NaCl) containing 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA, and the protease inhibitors aprotinin (25 g/ml), leupeptin (10 g/ml), and phenylmethylsulfonyl fluoride (1 mM). Co-immunoprecipitation Assay—Extracts in TBS/CHAPS buffer were clarified by centrifugation for 10 min at 15,000 ϫ g, and 1 ml aliquots of supernatant were transferred to a fresh 1.5-ml microcentrifuge tube This extract was incubated with 50 l of a 10% w/v solution of protein A-positive Staphylococcus aureus cells in 10% CHAPS lysis buffer for 30 min at 4 °C to remove cellular proteins binding to protein A. After washing two times in wash buffer (0.2% gelatin, 0.05% Tween 20 in TBS), blots were incubated for 1 h with horseradish peroxidase-labeled secondary antibody, washed again, and exposed to ECL color development reagents (Amersham Pharmacia Biotech)
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