Cytoskeletal Interactions with the Leukocyte Integrin β2 Cytoplasmic Tail

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 ␤2 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, ␤2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the ␤2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein ␣-actinin. The association of ␣-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. 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. Positive and inhibitory domains within the ␤2 tail regulate ␣-actinin binding: a single 11-amino acid region (residues 736 -746) is necessary and sufficient for ␣-actinin binding, and a regulatory domain between residues 748 -762 inhibits constitutive association of the ␤2 tail with ␣-actinin.Integrins are heterodimeric, transmembrane adhesion molecules composed of noncovalently associated ␣ and ␤ subunits that physically link extracellular ligands to the cytoskeleton (1). The cytoplasmic domain of integrin ␤ subunits links these receptors to the actin cytoskeleton (2, 3). However, actin filaments cannot bind directly to integrins. Instead, integrins are linked indirectly to actin filaments via several actin-binding proteins, including ␣-actinin, talin, and filamin. (4 -7). The importance of integrin-cytoskeletal linkage is demonstrated by the observation that deletion of the ␤ subunit tail prevents association of integrins with the cytoskeleton and disrupts normal integrin-ligand interactions (reviewed in Ref. 8).Several integrins, including LFA-1 1 and Mac-1, share a common ␤2 subunit and are present exclusively on leukocytes. These leukocyte integrins mediate cell adhesion to endothelial cell ligands such as intracellular adhesion molecules (reviewed in Ref. 9). Unactivated leukocytes in the circulation are nonadherent, and LFA-1 and Mac-1, both of which are expressed constitutively on resting neutrophils, show very little, if any, binding to their physiologic ligands (10). Activation of neutrophils with cytokines (leukotriene B 4 and tumor necrosis factor-␣), chemoattractants (FMLP), or phorbol 12-myristate 13-acetate (PMA) results in increased binding of neutrophils to the endothelium (11,12). This increase results, in part, from insertion of integrins onto the cell surface, but both LFA-1 and Mac-1 also undergo a rapid change in ligand avidity as a result of activation, which involves conformational changes...

A Single Amino Acid in the Cytoplasmic Domain of the β2 Integrin Lymphocyte Function-associated Antigen-1 Regulates Avidity-dependent Inside-out Signaling

“…Talin forms the bridge between the ␤ 2 integrin and the actin filaments. Upon activation, LFA-1 is released from the cytoskeleton as a result of proteolysis of talin, probably by calpain, leading to freely mobile integrin as postulated by Sampath et al (46). Next, ␣-actinin binds the ␤ 2 cytoplasmic domain between residues 736 and 746 directly C-terminal of the DLRE motif, thereby stabilizing the cytoskeleton-integrin interaction necessary for strong adhesion.…”

“…Calcium can activate specific proteases such as calpain that releases LFA-1 from the cytoskeleton (26). Presumably, talin is cleaved from the cytoskeleton (46), resulting in mobile LFA-1 and reorganization of the cytoskeleton network. Cytochalasin D disrupts the cytoskeleton and bypasses the PMA-induced activation.…”

The Cytoskeletal/Non-muscle Isoform of α-Actinin Is Phosphorylated on Its Actin-binding Domain by the Focal Adhesion Kinase

“…Tyrosines at positions 4,12,193, and 319 were replaced with phenylalanines using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) and Pfu Turbo DNA polymerase to make single base changes from TAT and TAC to TTT and TTC, respectively. The mutation primers for Tyr-4 were: 5Ј-ccg cgc acc atc atg gac cat ttt gat tct cag caa acc-3Ј (forward) and 5Ј-ggt ttg ctg aga atc aaa atg gtc cat gat ggt gcg cgg-3Ј (reverse), and those for Tyr-12 were 5Ј-gca aac caa cga ttt cat gca gcc aga aga gga ctg gg-3Ј (forward) and 5Ј-ccc agt cct ctt ctg gct gca tga aat cgt tgg ttt gc-3Ј (reverse).…”

“…The localization of ␣-actinin in focal adhesion plaques suggested that it might serve to anchor the network of actin filaments to the plasma membrane. This possibility was substantiated by the finding that ␣-actinin associates with the cytoplasmic tail of members of several adhesion receptors families including integrins (11,12), cadherins (13,14), and intercellular adhesion molecules (15,16). ␣-Actinin interacts with several cytoskeletal proteins in addition to actin.…”

Neutrophil integrin affinity regulation in adhesion, migration, and bacterial clearance

“…[14][15][16] Recently, it was shown that the protein level of gelsolin, an actin severing and capping protein, affects β 1 -integrin affinity and cell adhesion in the lymphocytic leukemia cell line L1210 and histiocytic lymphoma cell line U937. 17,18 As detected by 2D-gel electrophoresis, adherent growing L1210 cells (L1210-A) with active β 1 -integrins contained an almost 4-fold increase in gelsolin protein level compared with suspension growing L1210 cells (L1210-S) with inactive β 1 -integrins.…”