Cytoskeletal interactions with the leukocyte integrin beta2 cytoplasmic tail. Activation-dependent regulation of associations with talin and alpha-actinin

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.

Fig. 1. Interaction between the cytoplasmic domain of β2 and talin

A, co-immunoprecipitation of talin with β2 integrins. β2 integrins were immunoprecipitated from extracts of unactivated PMNs (−FMLP) or PMNs activated with 10 nM FMLP for 10 min, and these were immunoblotted with anti-talin. Intact (225 kDa) talin co-immunoprecipitates with β2 from unactivated cells (−FMLP) but not from activated cells (+FMLP). B, binding of purified talin to a GST-β2 cytoplasmic domain fusion protein. Purified talin was applied to GST fusion protein column expressing the entire β2 cytoplasmic domain (residues 724–769) or to a control column expressing GST only. Both intact, 225-kDa, talin and the 190-kDa proteolytic fragment of talin are present in the purified talin preparation. The intact, 225-kDa, talin band binds to the GST-β2 cytoplasmic domain fusion protein but does not bind to the GST alone. C, proteolysis of talin in PMNs following activation. Extracts of unactivated PMNs or PMNs activated with FMLP or PMA were immunoblotted with anti-talin. Talin from unactivated cells (−) was primarily (>90%) in the intact, 225-kDa form, with less than 10% in the 190-kDa form. Following activation with either FMLP or PMA, approximately 90% of the talin was in the 190-kDa form. D, calpeptin inhibits talin proteolysis in FMLP-activated PMNs. Pretreatment of PMNs with calpeptin (100 μM for 30 min) inhibited talin proteolysis.

Fig. 2. Association of α-actinin with β2 in PMNs requires activation of cells, whereas in fibroblasts, α-actinin associates constitutively with β1 integrins

An antibody against β2 was used to immunoprecipitate from extracts of PMNs under nondenaturing conditions that were unactivated (0 min) or were activated with FMLP (10 nM) for 5 or 10 min prior to extraction (left panel). Immunoprecipitates were immunoblotted with anti-α-actinin. Anti-β1 integrin was used to immunoprecipitate from extracts of rat embryo fibroblasts (REF-52) under nondenaturing conditions grown on either fibronectin or vitronectin or maintained in suspension for 2 h prior to extraction (right panel). α-Actinin co-precipitated with β1 integrins from adherent or suspension fibroblasts but co-precipitated with β2 integrins from PMNs only after activation.

Fig. 3. Analysis of α-actinin binding to GST-integrin cytoplasmic domain fusion proteins

A, comparison of α-actinin binding to β1 and β2 integrin cytoplasmic domain-GST fusion proteins. Cell extracts were applied to GST fusion protein affinity columns corresponding to integrin cytoplasmic tail constructs (illustrated in Table I), and eluates were immunoblotted with anti-α-actinin. α-Actinin bound relatively weakly to the full-length β2 cytoplasmic tail (β2 w.t.) but with relatively higher affinity to the full-length β1 cytoplasmic domain (β1 w.t.). B, comparison of α-actinin binding to distinct regions of the β₂ cytoplasmic domain. α-Actinin bound to the NH₂-terminal half (Δ746–769) but not to COOH-terminal half (Δ727–746) of the β2 tail. Deletion of residues 727–736 (Δ727–736) bound α-actinin weakly, at a level comparable to the full-length tail (β2-w.t.). C, confirmation of a binding site for α-actinin between residues 736 and 746. α-Actinin bound to a fusion protein containing only residues 736–746 (736–746). Control lanes confirm that α-actinin does not bind to the deletion mutant lacking residues 727–746 from the NH₂-terminal half of the tail (Δ727–746); α-actinin binds weakly to the wild-type β2 tail but binds with relatively higher affinity to the truncation mutant lacking the COOH-terminal half of the tail.

Fig. 4. Analysis of α-actinin binding to GST-β2 cytoplasmic domain fusion proteins with point mutations in the COOH-terminal half of the tail

Point mutations at residues Asn⁷⁴⁸, Asn⁷⁴⁹, Asp⁷⁵⁰, Pro⁷⁵², Thr⁷⁵⁸, Thr⁷⁵⁹, Thr⁷⁶⁰, Met⁷⁶² (not shown), and Ala⁷⁶⁷ each confer on the β2 tail the ability to bind α-actinin at levels comparable to that seen when the entire COOH-terminal half of the tail is deleted. Other point mutations of residues at the very end of the COOH terminus, Phe⁷⁶⁶ and Glu⁷⁶⁸, or the upstream lysine residue, Lys⁷⁵⁵, did not alter the wild-type (weak α-actinin binding) phenotype.

Fig. 5. A model to explain the role of talin and α-actinin in linking actin filaments to β2 integrins in resting and activated leukocytes

A, in unactivated cells, actin filaments are linked to integrins via their association with talin, which binds to the cytoplasmic domain of β2. Thus, in unactivated cells, mobility of β2 integrins is constrained by the actin cytoskeleton. Activation of cells results in proteolysis of talin, which causes talin to no longer bind to the β2 cytoplasmic domain, resulting in free mobility of the integrins in the membrane. This phase is transient, so that β2 integrins are rapidly reengaged by the actin cytoskeleton as a result of α-actinin binding to a previously cryptic binding site in the membrane proximal half of the cytoplasmic domain. B, the α-actinin binding domain in the β2 cytoplasmic tail (shown highlighted in green) is located between residues 736 and 746. The α-actinin inhibitory domain in the COOH-terminal half of the β2 tail (highlighted in red) prevents α-actinin binding in unactivated cells. Point mutations at several residues (residues shown in yellow) result in a mutant fusion protein that binds α-actinin with relatively higher affinity, suggesting that the conformation of the COOH-terminal half of the tail is critical for efficient inhibition of α-actinin binding to the membrane proximal half of the tail.