Among adhesion receptor families, integrins are particularly important in biological processes that require rapid modulation of adhesion and de-adhesion. Activation on a timescale of < 1 s of beta2 integrins on leukocytes and beta3 integrins on platelets enables deposition of these cells at sites of inflammation or vessel wall injury. Recent crystal, nuclear magnetic resonance (NMR), and electron microscope (EM) structures of integrins and their domains lead to a unifying mechanism of activation for both integrins that contain and those that lack an inserted (I) domain. The I domain adopts two alternative conformations, termed open and closed. In striking similarity to signaling G-proteins, rearrangement of a Mg2+-binding site is linked to large conformational movements in distant backbone regions. Mutations that stabilize a particular conformation show that the open conformation has high affinity for ligand, whereas the closed conformation has low affinity. Movement of the C-terminal alpha-helix 10 A down the side of the domain in the open conformation is sufficient to increase affinity at the distal ligand-binding site 9,000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. Recent structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the integrin headpiece, and a critical role for integrin epidermal growth factor (EGF) domains in the stalk region. The headpiece of the integrin faces down towards the membrane in the inactive conformation, and extends upward in a "switchblade"-like opening upon activation. These long-range structural rearrangements of the entire integrin molecule involving interdomain contacts appear closely linked to conformational changes within the I and I-like domains, which result in increased affinity and competence for ligand binding.
Icap1 alpha is a 200-amino acid protein that binds to the COOH-terminal 13 amino acids ((786)AVTTVVNPKYEGK(798)) of the integrin beta(1) subunit. Alanine scanning mutagenesis of this region revealed that Val(787), Val(790), and (792)NPKY(795) are critical for Icap1 alpha binding. The NPXY motif is a known binding substrate for phosphotyrosine binding (PTB) domain proteins. The sequences of Icap1 alpha, residues 58--200, and the beta(1) integrin, residues 786-797, were aligned to the available PTB-peptide structures to generate a high quality structural model. Site-directed mutagenesis showed that Leu(135), Ile(138), and Ile(139) of Icap1 alpha, residues predicted by the model to be in close proximity to (792)NPKY(795), and Leu(82) and Tyr(144), residues expected to form a hydrophobic pocket near Val(787), are required for the Icap1 alpha-beta(1) integrin interaction. These findings indicate that Icap1 alpha is a PTB domain protein, which recognizes the NPXY motif of beta(1) integrin. Furthermore, our date suggest that an interaction between Val(787) and the hydrophobic pocket created by Leu(82) and Tyr(144) of Icap1 alpha forms the basis for the specificity of Icap1 alpha for the beta(1) integrin subunit.
Integrins and other cell surface receptors have been fertile grounds for structure prediction experiments. Recently determined structures show remarkable successes, especially with beta-propeller domain predictions, and also reveal how ligand binding by integrins is conformationally regulated.
Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.
Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.
Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.
The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.
The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.
Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.
The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.
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.
Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.
Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single "fully competent" LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an alpha-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/DeltaD1-2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/DeltaD1-2 and with an approximately 2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/DeltaD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/DeltaD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, "fully competent" LFA-1-binding surface.
The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.
The cysteine-rich repeats in the stalk region of integrin beta subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the beta(2) subunit, one, KIM127, preferentially bound to alpha(L)beta(2) that was activated by mutations in the cytoplasmic domains, and by Mn(2+). KIM127 also bound preferentially to the free beta(2) subunit compared with resting alpha(L)beta(2). Activating beta(2) mutations also greatly enhanced binding of KIM127 to integrins alpha(M)beta(2) and alpha(X)beta(2). Thus, the KIM127 epitope is shielded by the alpha subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated alpha(L)beta(2) and bound equally well to resting and activated alpha(L)beta(2), differentially recognized resting alpha(M)beta(2) and alpha(X)beta(2), and bound fully to activated alpha(M)beta(2) and alpha(X)beta(2). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define five different beta(2) stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.
Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.
The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.
The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.
We introduced disulfide bonds to lock the integrin alphaLbeta2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing alphaLbeta2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated alphaLbeta2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal alpha-helix of the I domain, inhibited binding to ICAM-1 by alphaLbeta2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the alphaL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.