Cytoskeletal Interactions with the Leukocyte Integrin β2 Cytoplasmic Tail

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 β2 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, β2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the β2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein α-actinin. The association of α-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. Glutathione S-transferase-β2 tail fusion protein/mutagenesis experiments suggest that the affinity of α-actinin binding to the β2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic α-actinin binding domain. Positive and inhibitory domains within the β2 tail regulate α-actinin binding: a single 11-amino acid region (residues 736–746) is necessary and sufficient for α-actinin binding, and a regulatory domain between residues 748–762 inhibits constitutive association of the β2 tail with α-actinin. 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 β2 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, β2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the β2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein α-actinin. The association of α-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. Glutathione S-transferase-β2 tail fusion protein/mutagenesis experiments suggest that the affinity of α-actinin binding to the β2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic α-actinin binding domain. Positive and inhibitory domains within the β2 tail regulate α-actinin binding: a single 11-amino acid region (residues 736–746) is necessary and sufficient for α-actinin binding, and a regulatory domain between residues 748–762 inhibits constitutive association of the β2 tail with α-actinin. Integrins are heterodimeric, transmembrane adhesion molecules composed of noncovalently associated α and β subunits that physically link extracellular ligands to the cytoskeleton (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8988) Google Scholar). The cytoplasmic domain of integrin β subunits links these receptors to the actin cytoskeleton (2Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1648) Google Scholar, 3Pavalko F.M. Otey C.A. Proc. Soc. Exp. Biol. Med. 1994; 205: 282-293Crossref PubMed Scopus (99) Google Scholar). However, actin filaments cannot bind directly to integrins. Instead, integrins are linked indirectly to actin filaments via several actin-binding proteins, including α-actinin, talin, and filamin. (4Horwitz A.F. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (824) Google Scholar, 5Otey C.A. Pavalko F.M. Burridge K. J. Cell Biol. 1990; 111: 721-730Crossref PubMed Scopus (650) Google Scholar, 6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google Scholar, 7Sharma C.P. Ezzell R.M. Arnaout M.A. J. Immunol. 1995; 154: 3461-3470PubMed Google Scholar). The importance of integrin-cytoskeletal linkage is demonstrated by the observation that deletion of the β subunit tail prevents association of integrins with the cytoskeleton and disrupts normal integrin-ligand interactions (reviewed in Ref. 8Williams M.J. Hughes P.E. O'Toole T.E. Ginsberg M.H. Trends Cell Biol. 1994; 4: 109-112Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Several integrins, including LFA-1 1The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1; GST, glutathioneS-transferase; PMA, phorbol 12-myristate 13-acetate; FMLP, formyl-methionyl-leucyl-phenylalanine; PMN, polymorphonuclear leukocyte; PBS, phosphate-buffered saline; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate; w.t., wild-type; TBS, Tris-buffered saline. 1The abbreviations used are: LFA-1, lymphocyte function-associated antigen-1; GST, glutathioneS-transferase; PMA, phorbol 12-myristate 13-acetate; FMLP, formyl-methionyl-leucyl-phenylalanine; PMN, polymorphonuclear leukocyte; PBS, phosphate-buffered saline; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate; w.t., wild-type; TBS, Tris-buffered saline. and Mac-1, share a common β2 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. 9Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5845) Google Scholar). 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 (10Smith C.W. Marlin S.D. Rothlein R. Toman C. Anderson D.C. J. Clin Invest. 1989; 83: 2008-2017Crossref PubMed Scopus (952) Google Scholar). Activation of neutrophils with cytokines (leukotriene B4 and tumor necrosis factor-α), chemoattractants (FMLP), or phorbol 12-myristate 13-acetate (PMA) results in increased binding of neutrophils to the endothelium (11Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1286) Google Scholar, 12Peter K. O'Toole T.E. J. Exp. Med. 1995; 181: 315-326Crossref PubMed Scopus (145) Google Scholar). 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 in their extracellular domain and a clustering of the receptors in the membrane. The association of cytoskeletal proteins with integrin cytoplasmic tails appears to play an important role in regulating intracellular signaling events that affect the conformation of integrin extracellular domains and promote integrin clustering. Several recent studies suggest that LFA-1 on lymphocytes is constrained by the actin cytoskeleton in resting cells (13Stewart M.P. McKowall A. Hogg N. J. Cell Biol. 1998; 140: 699-707Crossref PubMed Scopus (276) Google Scholar, 14Lub M. van Vliet S.J. Oomen S. Pieters R.A. Robinson M. Figdor C.G. van Kooyk Y. Mol. Biol. Cell. 1997; 8: 719-728Crossref PubMed Scopus (46) Google Scholar, 15Kucik D.F. Dustin M.L. Miller J.M. Brown E.J. J. Clin. Invest. 1996; 97: 2139-2144Crossref PubMed Scopus (294) Google Scholar) and that Ca2+-mediated activation of the enzyme calpain releases LFA-1 from this constraint to allow integrin clustering upon activation (14Lub M. van Vliet S.J. Oomen S. Pieters R.A. Robinson M. Figdor C.G. van Kooyk Y. Mol. Biol. Cell. 1997; 8: 719-728Crossref PubMed Scopus (46) Google Scholar). Kucik et al. (15Kucik D.F. Dustin M.L. Miller J.M. Brown E.J. J. Clin. Invest. 1996; 97: 2139-2144Crossref PubMed Scopus (294) Google Scholar) also demonstrated that the mobility of LFA-1 in lymphocytes is increased by activation with phorbol ester. In this report, we examine the association of two cytoskeletal proteins that directly interact with β1 and β2 integrin cytoplasmic domains to learn more about how these associations may be regulated. Based on our results, we propose a model in which β2 integrins on unactivated PMNs are tethered to the actin cytoskeleton via the protein talin. Following activation, proteolysis of talin causes release of integrins from the actin cytoskeleton. This would facilitate increased integrin mobility in the membrane, increased ligand binding, and clustering of ligand occupied receptors. Following clustering, integrins reengage the cytoskeleton by binding to the actin cross-linking protein α-actinin as we have previously described (6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google Scholar). Results of the glutathioneS-transferase (GST) fusion protein affinity binding experiments presented here suggest that the association of α-actinin with β2 integrins is regulated by conformational changes in the cytoplasmic domain that unmask the α-actinin binding site between residues 736 and 746 in the membrane proximal half of the tail that is cryptic resting cells. Our model helps explain how the cytoskeleton plays an apparent dual role in the regulation of integrin-mediated leukocyte adhesion: inhibition of adhesion in resting cells but stimulation of adhesion and motility in activated cells. 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). The PMN pellet, which also contained monocytes, was washed in Dulbecco's phosphate buffered saline (PBS) containing 0.2% glucose and resuspended in Dulbecco's PBS with glucose at a concentration of 107 cells/ml. PMNs were activated with either 10 nmformyl-methionyl-leucyl-phenylalanine (FMLP) peptide (Sigma) or 50 nm PMA (Sigma). In some experiments, PMNs were preincubated in the calpain inhibitor calpeptin (Calbiochem-Novabiochem), which was prepared in Me2SO at 100 mm and diluted to a final concentration of 100 μm (36 μg/ml) in Dulbecco's PBS. Control cells were incubated in Dulbecco's PBS containing 1 μl of Me2SO/ml. 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 μg/ml), leupeptin (10 μg/ml), and phenylmethylsulfonyl fluoride (1 mm). Following extraction for 10 min on ice, insoluble material was removed by centrifugation at 14,000 × g, and the supernatant was saved. Protein concentration was determined using BCA reagent (Pierce); approximately 20 μg was loaded per lane for immunoblot analysis, and 500 μg was loaded onto GST fusion protein affinity columns. For co-immunoprecipitation experiments, 5 × 106 cells were lysed in 1 ml of TBS/CHAPS buffer containing 1% CHAPS, 10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 0.01% NaN3, and 20 mm DNase I and protease inhibitors aprotinin (25 μg/ml), leupeptin (10 μg/ml), and phenylmethylsulfonyl fluoride (1 mm) as described previously (6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google Scholar). Rat embryo fibroblasts (REF-52) were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C in 5% CO2/95% air in a humidified incubator. For co-immunoprecipitation experiments, cells were plated on vitronectin-coated (10 μg/ml) or fibronectin-coated (50 μg/ml) plastic in Dulbecco's modified Eagle's medium without serum for 6 h. Suspension cells were obtained by trypsinization of substrate adherent cells, which were then washed in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and maintained in suspension at 37 °C for 2 h to allow the cells time to recover. Extracts in TBS/CHAPS buffer were clarified by centrifugation for 10 min at 15,000 ×g, and 1 ml aliquots of supernatant were transferred to a fresh 1.5-ml microcentrifuge tube. This extract was incubated with 50 μl 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. The S. aureuscells were sedimented, and an excess of either anti-β2 mAb (KIM127 or MHM23, Dako, Inc.) or anti-β1 integrin antibody (1938, Chemicon) was added to the supernatant and then incubated for 1 h at 4 °C. Then, 100 μl of a 10% suspension of protein A-Sepharose conjugated to rabbit anti-mouse Ig was added to the mixture, which was incubated for an additional 1 h at 4 °C. The complexes containing antibody-bound integrin and proteins were and washed with TBS/CHAPS buffer containing Then, μl of buffer was added to the which were to release were on 10% and transferred to or α-actinin with integrins was using 8d4 or from and the the β2 or β1 integrin cytoplasmic tails were by and A. The region the cytoplasmic tails of these integrins was by and the GST fusion cytoplasmic domain were by using the of T.A. Proc. S. A. PubMed Scopus Google Scholar). The were by of the region of The fusion protein were the and of the or proteins was with GST fusion proteins were using on and then to using for 1 h at to in affinity as described by the 500 μg of PMN or 5 μg of α-actinin or talin was to the and to bind for 30 min and talin were from as described previously C.A. Pavalko F.M. Burridge K. J. Cell Biol. 1990; 111: 721-730Crossref PubMed Scopus (650) Google Scholar). proteins were removed by with cell lysis and proteins were with 1 containing 10% mm EDTA, and 10 mm Following acid and were to and transferred to analysis were by for 1 h in buffer containing serum 0.2% and 20 in and with antibody or anti-talin antibody for 1 h. two in buffer 20 in were incubated for 1 h with washed and to We previously that the protein α-actinin associated with the cytoplasmic domain of the β2 subunit in PMNs following activation but that α-actinin interact with β2 integrins in unactivated cells (6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google Scholar). we for cytoskeletal proteins that constitutively to β2 integrins and to link integrins to actin filaments to activation. For these experiments, we used an integrin co-immunoprecipitation We that the protein talin, previously to bind to the cytoplasmic domain of β1 (4Horwitz A.F. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (824) Google Scholar) and integrins J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google with β2 integrins from unactivated PMNs 1 the of talin was in the β2 from unactivated the of talin, present in PMN was present in the integrin talin to with β2 integrins activation 1 the talin, to the to constitutively bind to the cytoplasmic tail. The in the two that the talin and the to the Ig to be an additional of talin. In to we have also that talin from bind directly to the β2 cytoplasmic tail. In this the talin the to a GST fusion protein to the β2 integrin cytoplasmic tail but bind to GST 1 we the of the observation that talin with β2 integrins from resting that talin proteolysis in PMNs following activation was by immunoblot 20 μg of protein from unactivated PMNs or μg of protein from PMNs activated with either the peptide (10 or with the phorbol PMA was by transferred to and to immunoblot analysis using a antibody talin of protein from and cells was necessary to allow to the of the talin that was present following 1 that in resting of the talin is in the to the The of the to the were determined by to and following activation of PMNs with or In unactivated cells, the of talin to was following activation, the of talin to was 1 that of PMNs for 30 min with the membrane calpain inhibitor calpeptin talin proteolysis following activation that of the talin to the of these results suggest that talin with the β2 cytoplasmic tail as determined by co-immunoprecipitation Following activation, the of the talin is by a protease to a that with the β2 tail. with our (6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google we that association of α-actinin with β2 integrins in PMNs activation of the cells. 2 that following activation, α-actinin with the cytoplasmic tail and be in of β2 In α-actinin in fibroblasts appears to be constitutively associated with the β1 integrins. in α-actinin be by in with anti-β1 antibody from fibroblasts that have either grown on or or maintained in suspension for 2 h. We that between the β1 and β2 cytoplasmic domains may be for regulating this association of α-actinin. this we GST fusion proteins to that to the β1 or β2 cytoplasmic domains residues residues and used as affinity to the of α-actinin from cell extracts to bind to and I show that α-actinin to the β1 tail but with the β2 tail fusion binding to the β1 tail was binding to the β2 tail results were obtained α-actinin, of cell was to the fusion protein affinity with the for activation of PMNs to binding, these results suggest that additional such as conformational changes in the β2 are to allow binding of α-actinin to the cytoplasmic of binding acid increase in α-actinin to β2 w.t., β2 cytoplasmic are by are β2 w.t., β2 cytoplasmic domain. in a are by are with this to remove the half of the β2 cytoplasmic tail at 746 in binding of α-actinin with the β2 tail and Results from experiments that α-actinin binding to with α-actinin binding to The membrane proximal half of the cytoplasmic domain of β2 expressed in the several including the that was previously in binding (6Pavalko F.M. LaRoche S.M. J. Immunol. 1993; 151: 3795-3807PubMed Google Scholar). with the importance of residues for binding, and I also show that α-actinin be binding to a deletion in which to the membrane proximal half of the tail were deletion residues α-actinin with an increase of β2 binding and I show that the 11-amino acid region within residues is sufficient for binding α-actinin at a β2 and was both to α-actinin binding to and to the β2 the membrane proximal These results that a binding site for α-actinin within residues and that binding of α-actinin to this region may be or by an inhibitory domain between residues the α-actinin inhibitory domain in the membrane half of the β2 cytoplasmic tail regulate integrin association with α-actinin, a of were in this The results of experiments the of α-actinin to bind to these fusion proteins are in in at residues in the of the 4 and and in binding of α-actinin at to or α-actinin binding to at including and in 4 and however, the binding binding of α-actinin. the conformation of the half of the β2 cytoplasmic domain by of at of several residues the inhibitory on α-actinin binding to residues in the membrane proximal half of the tail. to be a role for the of the between residues and in regulating cytoskeletal the of the that α-actinin binding is The cytoplasmic tails of integrin β subunits play an important role in interactions with the cytoskeleton and are important in and Integrins that are in the membrane and physically with cytoskeletal proteins in and in F.M. Otey C.A. Proc. Soc. Exp. Biol. Med. 1994; 205: 282-293Crossref PubMed Scopus (99) Google Scholar, C.A. Burridge K. J. Biol. 1993; Full Text PDF PubMed Google Scholar). In this we have the role of the β2 integrin cytoplasmic tail in interactions with two cytoskeletal proteins, talin and α-actinin, using neutrophils and cytoplasmic domain fusion protein affinity columns. Based on our results, we propose a mechanism in which β2 integrins in unactivated neutrophils are tethered to the actin cytoskeleton a linkage mediated by talin. The proteolysis of talin, which be by the calpain protease inhibitor may regulate binding of talin to β2 integrins. a role for talin in lymphocytes to explain the regulation of adhesion by calpain (13Stewart M.P. McKowall A. Hogg N. J. Cell Biol. 1998; 140: 699-707Crossref PubMed Scopus (276) Google Scholar). et al. (14Lub M. van Vliet S.J. Oomen S. Pieters R.A. Robinson M. Figdor C.G. van Kooyk Y. Mol. Biol. Cell. 1997; 8: 719-728Crossref PubMed Scopus (46) Google Scholar) that the cytoskeleton on adhesion on or LFA-1 was or was on the membrane. that release of cytoskeletal on LFA-1 in unactivated lymphocytes was necessary to allow of LFA-1, and clustering on the membrane was necessary to stabilize cell Following activation, β2 integrins may be from cytoskeletal constraint by the link between talin and integrin upon proteolysis of talin. β2 integrins have ligand and in the membrane, of the actin mediated by binding of α-actinin to the membrane proximal half of the may stabilize interactions necessary for firm adhesion and domains within the β2 cytoplasmic tail that regulate binding to the protein α-actinin have also cytoplasmic tail fusion protein binding experiments an 11-amino acid region between residues 736 and 746 in the membrane proximal half of the tail that are necessary and sufficient for α-actinin region in the membrane half of the tail was to have a role in regulating α-actinin inhibitory region between residues and prevents association of α-actinin to the membrane proximal binding The of at of several residues in this inhibitory domain (residues and to unmask the previously cryptic α-actinin binding site in the β2 tail suggest that the conformation of the tail is important in regulating α-actinin of the membrane region (residues the of several within this domain that have on the of the β2 cytoplasmic tail. of these is the in which is is the integrins β subunits T.A. Protein 1998; Scopus Google and to be important for such as cytoskeletal firm and change C. J. J. Cell Biol. 1997; PubMed Scopus Google Scholar, T.E. J. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with integrin in and in to on vitronectin-coated the domain was by cells with and E.J. C. J. Cell Biol. 1995; PubMed Scopus Google Scholar). This deletion to affect ligand the cells were to bind et al. M.L. S. Springer T.A. J. Exp. Med. PubMed Scopus Google Scholar, M.L. Springer T.A. PubMed Scopus Google Scholar) have also that within the of β2 binding of cells to intracellular adhesion surface, of a This that this have a regulatory role both in protein conformation and in a site for also a β to the cytoplasmic domain. Our model a of the β2 tail in unactivated neutrophils in which the α-actinin binding site is as a result of this This model is by the of et al. A. A.F. J. Cell Biol. 1992; PubMed Scopus Google that of the in the region of the domain in β1 integrins that were for cytoskeletal binding were in on of an The mechanism of conformational change in however, is The membrane half of the β2 tail several including a and the al. M.L. S. Springer T.A. J. Exp. Med. PubMed Scopus Google Scholar, M.L. Springer T.A. PubMed Scopus Google Scholar) have that in of the β2 tail was at of this by on ligand of the to however, to a of ligand binding in their that the region between and be important for ligand binding and association of cytoskeletal In our which may by a and both α-actinin binding with that of is necessary for binding of α-actinin but may be important for ligand of the previously to be important for extracellular ligand binding the cytoskeletal protein binding described here in suggest that domains in the β2 cytoplasmic tail regulate these two et al. M.L. S. Springer T.A. J. Exp. Med. PubMed Scopus Google Scholar, M.L. Springer T.A. PubMed Scopus Google Scholar) that the region between and in the β2 tail was for intracellular adhesion binding, we have α-actinin binding to this region and have demonstrated the for of for ligand binding, we have that single at of induces α-actinin the acid have in ligand binding, in the In our studies that maintained the β2 tail in a was to bind of residues important for cytoskeletal association adhesion to in regulating the association with in regulating binding M.L. Springer T.A. PubMed Scopus Google the inhibitory the inhibitory on our and of et al. M.L. Springer T.A. PubMed Scopus Google is that within the β2 tail are in regulating intracellular association with α-actinin and extracellular association with in a Based on our and of et al. M.L. Springer T.A. PubMed Scopus Google is that within the β2 tail are in regulating intracellular association with α-actinin and extracellular association with these studies suggest of some as cytoplasmic which directly regulate both the that proteins, such as S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google interact with β2 integrin cytoplasmic tail and regulate proteins that are for β1 and have also described C. J. S. Nature. 1996; PubMed Scopus Google Scholar, S.J. O'Toole T.E. M. M. Ginsberg M.H. J. Cell Biol. 1995; PubMed Scopus (164) Google Scholar) that to interact with β integrins the of in their cytoplasmic The interaction of these proteins with integrins to in their that explain regulation of the et al. (14Lub M. van Vliet S.J. Oomen S. Pieters R.A. Robinson M. Figdor C.G. van Kooyk Y. Mol. Biol. Cell. 1997; 8: 719-728Crossref PubMed Scopus (46) Google Scholar) have demonstrated of signaling for activation of β1 and via their cytoplasmic and have the for of lymphocyte in β2 integrin activation. at proteins that interact with the β2 tail in the of our here important of activation. We for and for in the of GST fusion