Urokinase-type plasminogen activator receptor induces conformational changes in the integrin alphaMbeta2 headpiece and reorientation of its transmembrane domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.     

Identification of two molecular defects in a child with leukocyte adherence deficiency.

Children with leukocyte adherence deficiency (LAD), or leukocyte cell adhesion molecule deficiency, experience recurrent, life-threatening bacterial infections related to severe deficiency in surface expression of the leukocyte integrin molecules. The leukocyte integrins consist of a common CD18 (beta) subunit and individual, noncovalently associated alpha subunits designated CD11a, CD11b, and CD11c. Defects in the CD18 subunit prevent surface expression of the CD11/CD18 complexes in children with this disease. We investigated the molecular basis of the disease in a child with the severe deficiency form of LAD and identified two molecular defects in the CD18 subunit. The first defect is a single-base pair C----T transposition resulting in an amino acid substitution of a leucine for a proline at amino acid 178. This amino acid substitution is located in a region that is highly conserved among the integrin beta subunits and where two previous defects have been located in LAD. The second mutation involves a deletion of 220 base pairs in the cDNA coding for a portion of the extracellular domain and results in a frameshift into a premature stop codon. The deleted region corresponds to a single exon in the CD18 gene. Identification of these two molecular defects in a single child with this disease indicates the compound heterozygous nature of the disorder in this child and identifies regions of the CD18 subunit that may be important for CD11/CD18 heterodimer formation and surface expression.     

Characterization of macrophage adhesion molecule

Macrophage adhesion molecule (MAM), an abundant surface molecule which functions in the adhesion and spreading of guinea pig macrophages on surfaces, is characterized as a heterodimer of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). The density of MAM molecules is estimated at 630,000 per macrophage on the basis of quantitative binding of 125I-labeled monoclonal antibody. The glycopeptide subunits display microheterogeneity on isoelectrofocusing; the pI is 5.8-6.3 for gp160 (MAM-alpha) and 6.4-7.0 for gp93 (MAM-beta). A neutrophil gp160, gp93 molecule was shown to be indistinguishable from macrophage MAM on the basis of electrophoresis, isoelectrofocusing, and reactivity with 10 monoclonal antibodies. A related heterodimer of gp93 associated with a larger, antigenically different glycopeptide (gp180,gp93) was identified on circulating lymphocytes. Cumulative properties indicate that MAM is the guinea pig analogue of human Mo1 and mouse Mac-1.     

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent,but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.     

Genetic analysis of integrin function in man: LAD-1 and other syndromes.

The integrins are cell membrane receptors composed of alpha and beta subunits which orchestrate adhesive events in all tissues of the body (Hynes, R.O., 1992. Integrins: versatility, modulation, and signalling in cell adhesion. Cell 69, 11-25; and Hynes, R.O., 1999. Cell adhesion: old and new questions. Trends Cell Biol. 9, M33-37). At present 18 alpha subunits and 8 beta subunits have been identified which are loosely organised into families. There are three inherited autosomal recessive diseases in man which involve germline mutations in genes coding for integrins. Leukocyte adhesion deficiency-1 (LAD-1) is the result of mutations in the beta2 subunit of the CD11/CD18 integrins, LFA-1, Mac-1, p150,95 and alphadbeta2. The bleeding disorder Glanzmann thrombasthenia is caused by mutations in either the alpha or beta subunit of the platelet integrin, alphaIIbbeta3. Thirdly, it is now recognised than one of the variants of the usually lethal skin blistering disorder, epidermolysis bullosa (JEB-PA), is caused by mutation in either the alpha or beta subunit of the epithelial hemidesmosome integrin, alpha6beta4. Many of the mutations cause defective alphabeta heterodimer formation. The majority of the beta subunit mutations are in the conserved N-terminal region known as the betaI domain. It is suggested that this region participates in alphabeta heterodimer formation.     

Adhesion of neutrophils to fibronectin: role of the cd66 antigens

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Mouse M290 is the functional homologue of the human mucosal lymphocyte integrin HML-1: antagonism between the integrin ligands E-cadherin and RGD tripeptide.

Human mucosal lymphocyte antigen-1 (HML-1, alphaEbeta7) and E-cadherin, two members of unrelated cell adhesion superfamilies, have evolved to play cooperative roles in gut mucosal immunity. Human E-cadherin is self-ligand mediating intercellular adhesion of epithelial cells, as well as adhesion of intra-epithelial lymphocytes to intestinal enterocytes via an interaction with HML-1. Herein we report that both dimeric and monomeric forms of recombinant mouse E-cadherin-human immunoglobulin Fc chimera self-associate and support attachment of E-cadherin+ mouse colon epithelial cells. Both forms also support the adhesion of mouse MTC-1 T cells via M290, thereby establishing M290 as the functional mouse homologue of HML-1 and revealing that E-cadherin homophilic and heterophilic binding sites are distinct. Adhesion of MTC-1 cells to E-cadherin-Fc was inhibited by arginine-glycine-aspartate (RGD) peptides and vice versa cells bound to immobilized RGD polymer in an M290-dependent fashion, where adhesion was inhibitable with soluble E-cadherin-Fc. Hence, E-cadherin and RGD integrin ligands antagonize cell binding by one another, either by inducing integrin cross-talk or by binding to shared or overlapping sites within M290. Binding of E-cadherin-Fc by HML-1 costimulated the CD3-induced proliferation of purified CD4+ T cells, suggesting that E-cadherin expressed on dendritic cells may play a T cell costimulatory role in addition to facilitating dendritic cell-keratinocyte adhesion.     

A naturally occurring extracellular alpha-beta clasp contributes to stabilization of beta3 integrins in a bent, resting conformation

Control of alphaIIb beta3 and alphav beta3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin alpha and beta subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or "off" conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of alphav beta3. In this bent structure, a segment of alphav (301-308) and beta3 (560-567) are juxtaposed. Here we provide evidence that these regions of alphav/alphaIIb and beta3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the alphaIIb and beta3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three beta3 LIBS antibodies. Mutation of the clasp region of alphav or beta3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the alphav beta3 structure yield a refined model for the alphav beta3 clasp and provide plausible explanations for the effects of the activating mutations.     

Modulation of CD11b/CD18 adhesive activity by its extracellular,membrane-proximal regions

The integrin receptor CD11b/CD18 is normally kept in a low adhesive state and can be activated by many different agents. However, the mechanism underlying receptor activation is not yet fully understood. We hypothesized that the extracellular, membrane-proximal regions of CD11b/CD18 are critically involved in modulation of its adhesive functions. To test our hypothesis, we perturbed the extracellular, membrane-proximal regions of individual CD11b and CD18 subunits and studied their effect on ligand binding, receptor clustering, and lipid raft association. We report here three major findings: 1) perturbation of the extracellular, membrane-proximal region of either subunit leads to enhanced adhesion, caused by changes in receptor conformation, but not the state of receptor clustering or lipid raft association; 2) the CD11b subunit plays a more important role in confining the receptor in an inactive state; and 3) upon modification of the extracellular, membrane-proximal region, the mutant CD11b/CD18 acquires the ability to respond to stimulation by "inside-out" signaling. Our results suggest that the extracellular, membrane-proximal region of the receptor plays an important role in integrin activation and therefore could be targeted by certain cell surface proteins as a conduit to control the integrin "inside-out" signaling process.     

Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

JAM-L–mediated leukocyte adhesion to endothelial cells is regulated in cis by α4β1 integrin activation

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L–VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.     

The I domain is a major recognition site on the leukocyte integrin Mac- 1 (CD11b/CD18) for four distinct adhesion ligands

Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-terminal and perhaps divalent cation binding regions but not the COOH-terminal segment may contribute. The recognition sites in the I domain appear overlapping but not identical as individual Mac-1-ligand interactions are distinguished by the discrete patterns of inhibitory mAbs. Additionally, we find that the alpha subunit NH2-terminal region and divalent cation binding region, despite being separated by over 200 amino acids of the I domain, appear structurally apposed because three mAbs require the presence of both of these regions for antigenic reactivity, and chimeras that contain the NH2 terminus of p150,95 require the divalent cation binding region of p150,95 to associate firmly with the beta subunit.     

Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.     

Involvement of the CD11b/CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity.

TNF-alpha can incite neutrophil-mediated endothelial cell damage and neutrophil H2O2 release. Both effects require adherent neutrophils. Using specific mAb, we showed in this in vitro study that the CD18 beta 2-chain and the CD11b alpha M-chain of the CD11/CD18 integrin heterodimer have a major role in both TNF-alpha-induced neutrophil-mediated detachment of human umbilical vein endothelial cells and H2O2 release by TNF-alpha-activated human neutrophils. In contrast to anti-CD18 mAb, which consistently prevented neutrophil activation, anti-CD11a mAb and two of three anti-CD11b mAb did not reduce endothelial cell detachment and neutrophil H2O2 release, although they decreased neutrophil adhesion to human umbilical vein endothelial cells. mAb 904, directed against the bacterial LPS binding region of CD11b, reduced endothelial cell detachment for about 40% and neutrophil H2O2 release for more than 50%, demonstrating that CD11b/CD18 is engaged in TNF-induced neutrophil activation. Dependence on CD11b/CD18 could not be overcome by CD18-independent anchoring of neutrophils via PHA. Additionally, neither induction of increased expression of the endothelial cell adhesion molecules ICAM-1 and ELAM-1, nor subsequent addition of specific mAb, influenced endothelial cell injury or H2O2 release by TNF-activated neutrophils. Interaction with ICAM-1 and ELAM-1 therefore appears not to induce additional activation of TNF-stimulated neutrophils. These studies suggest that a specific, CD11b/CD18-mediated signal, instead of adherence only, triggers toxicity of TNF-activated neutrophils.     

CD9-alpha6beta1 interactions in migratory parietal endoderm cells

Tetraspanins modulate the function of a variety of membrane proteins, including integrin receptors. We show here that the tetraspanin CD9 preferentially coimmunoprecipitates with the alpha6beta1 integrin heterodimer in F9-derived parietal endoderm cells in comparison to F9 stem cells. We also show that CD9 function-blocking antibody inhibits parietal endoderm migration in an embryoid body outgrowth assay. In addition, both CD9 and alpha6beta1 colocalize with vinculin to apparent focal adhesion sites in parietal endoderm cells. The data presented here suggests a role for CD9 in localizing the integrin to the focal adhesion. In addition, the data suggest a role for CD9 in alpha6beta1 mediated migration of parietal endoderm.     

Purification, composition, and structure of macrophage adhesion molecule

Macrophage adhesion molecule (MAM) is a surface heterodimer consisting of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). MAM, which is the guinea pig analogue of Mo1 and Mac-1, was purified from detergent lysates of peritoneal neutrophils by lentil lectin chromatography and M2-antibody chromatography. The pure heterodimer molecule was dissociated by acidic conditions (pH 3.5), and MAM-alpha and MAM-beta were separated by M7-antibody chromatography. MAM-beta is an approximately 640 amino acid residue polypeptide with exceptionally high cysteine content. At 7.2 residues per 100 amino acids, Cys/2 of MAM-beta is more than 3 times the mean for 200 purified proteins. Reactivity with six beta-subunit-specific monoclonal antibodies recognizing at least four epitopes demonstrated that intrapeptide disulfide bonds are required to maintain the structure of MAM-beta. All six antibodies failed to react when MAM-beta was treated with reducing agents. MAM-beta is 18% carbohydrate; the major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid. MAM-beta is estimated to contain five to six N-linked carbohydrate units. MAM-alpha is an approximately 1100-residue polypeptide with lower Cys/2 content (2.0 residues per 100 amino acid residues). MAM-alpha is 21% carbohydrate. The major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid; the mannose content is higher in MAM-alpha than MAM-beta. MAM-alpha is estimated to contain 12 N-linked carbohydrate units.     

Up-regulation of VCAM-1 and differential expansion of beta integrin-expressing T lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis infection

Immune responses rely on an intricate system of adhesion molecules to coordinate the homing and retention of lymphocytes in both secondary lymphoid tissues and at sites of infection. To define the events associated with pulmonary immune responses, the expression of endothelial addressins and integrins on T cells was analyzed during Mycobacterium tuberculosis infection. In infected lung, expression of endothelial VCAM-1, but not mucosal addressin cell adhesion molecule-1, was up-regulated from 4 wk postinfection and persisted to at least 12 wk. Subsequent analysis of the corresponding integrins expressed on lung CD4+ and CD8+ T cells revealed an accumulation of beta1high/beta7-/low, and to a lesser extent beta7high, integrin-expressing T cells during infection. Examination of integrin heterodimers showed that while alpha4 integrin was predominantly expressed on beta1high/beta7-/low cells, alphaE integrin was primarily associated with beta7high. The majority of activated/memory T cells recruited during infection expressed high levels of beta1 integrin and undetectable or low levels of beta7 integrin. These T cells were capable of producing IFN-gamma, a cytokine crucial for controlling M. tuberculosis infection. Rapid expansion of beta1high, beta7-, and beta7high T cell populations in the lung upon secondary mycobacterial infection indicates the participation of these populations in the acquired immune response to the infection. Furthermore, treatment of infected mice with mAb to alpha4 or alpha4beta7 integrin led to a reduction in lymphocytes and increase in granulocytes in the pulmonary infiltrate. These results reveal a crucial role for adhesion molecules in the generation of an effective pulmonary immune response to M. tuberculosis infection.     

C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1

Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by 'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.