Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.     

Structure-function of the putative I-domain within the integrin beta 2 subunit

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 microm), which is most likely located within this region of beta(2).     

Loops within the CD11c I domain critical for specific recognition of fibrinogen

The I domains of CD11 are responsible for the binding of ligands and have a unique structure with 6-7 alpha helices and 6 beta sheets with interconnecting loops. To determine loops recognizing fibrinogen in CD11c I domain, five oligopeptides corresponding to CD11c loops were used to prevent fibrinogen binding to isolated CD11c I domain. The results of the inhibition experiment indicated that all of the loops except the one between E-beta sheet and 6-alpha helix were involved in the binding to fibrinogen. The peptide beta D alpha 5 and alpha 3 alpha 4 showed higher inhibitory activity than others, and the combination of four peptides blocked fibrinogen binding to the I domain completely. These peptides (beta A alpha 1, alpha 3 alpha 4, beta D alpha 5 and beta F alpha 7) could block THP-1 cell binding to fibrinogen coated surface as well. Alanine substitution of amino acids on the I domain such as Y249A and Q201A (which are on the loops beta D-alpha 5 and alpha 3-alpha 4 respectively) abolished fibrinogen binding, while mutation on the loop beta E-alpha 6 (Q273A) had no effect on fibrinogen binding. Taken together, the results from this study suggest that the loops on the top of CD11c I domain such as loop beta A-alpha 1, alpha 3-alpha 4, beta D-alpha 5 and beta F-alpha 7 are involved in fibrinogen binding, and two loops (alpha 3-alpha 4 and beta D-alpha 5) are more important than others for the recognition of fibrinogen.     

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.     

Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.

Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

The fourth blade within the beta-propeller is involved specifically in C3bi recognition by integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha M beta 2 are critical to phagocytosis of opsonized particles in host defense against foreign pathogens and certain malignant cells. Previous studies have mapped critical residues for C3bi binding to the I-domains of the alpha M and the beta2 subunits. However, the role of the alpha M beta-propeller in ligand binding remains less well defined, and the functional residues are still unknown. In the present study, we studied the function of the alpha M beta-propeller in specific ligand recognition by alpha M beta 2 using a number of different approaches, and we report four major findings. 1) Substitution of five individual segments (Asp398-Ala402, Leu412-Leu419, Tyr426-Met434, Phe435-Glu443, and Ser444-Thr451) within the W4 blade of the beta-propeller with their homologous counterparts in integrin alpha2 abrogated C3bi binding, whereas substitution of eight other segments outside this blade had no effect. 2) These five mutants defective in C3bi binding supported strong alpha M beta 2-mediated and cation-dependent cell adhesion to fibrinogen, suggesting that the conformations of these five defective mutants were intact. 3) Polyclonal antibodies recognizing sequences within the W4 blade significantly blocked C3bi binding by wild-type alpha M beta 2. 4) A synthetic peptide corresponding to Gln424-Gly440 within W4 interacted directly with C3bi. In conclusion, our data demonstrate that the W4 blade (residues Asp398 to Thr451) is involved specifically in C3bi but not fibrinogen binding to alpha M beta 2. Altogether, our study supports a model in which three separate domains of alpha M beta 2 (the alpha MI-domain, the alpha M beta-propeller, and the beta 2I-domain) function together and contribute to the formation of the C3bi-binding site.     

The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1)

The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.     

A molecular basis for integrin alphaMbeta 2 ligand binding promiscuity

The leukocyte integrin alpha(M)beta(2) is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of alpha(M)beta(2), the inter-integrin chimera was created. In the chimeric integrin, the betad-alpha5 loop-alpha5 helix segment comprised of residues Lys(245)-Arg(261) from the alpha(M)I domain of alpha(M)beta(2) was inserted into the framework of alpha(L)beta(2). The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the alpha(M)(Lys(245)-Arg(261)) sequence converted alpha(L)beta(2) into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type alpha(M)beta(2). Verifying a switch in the binding specificity of alpha(L)beta(2), the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe(246), Asp(254), and Pro(257) within Lys(245)-Arg(261) of alpha(M)beta(2) produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of alpha(M)(Lys(245)-Arg(261)) imparted to the chimeric integrin the ability to recognize many typical alpha(M)beta(2) protein ligands. Furthermore, cells expressing the chimeric receptor, but not alpha(L)beta(2), were able to stick to uncoated plastic, which represents the hallmark of wild-type alpha(M)beta(2). These results suggest that alpha(M)(Lys(245)-Arg(261)) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to alpha(M)beta(2).     

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.     

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.     

Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state

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.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Tests of the extension and deadbolt models of integrin activation

Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.     

Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues for alphaIIbeta3 binding.

The red cell intercellular adhesion molecule-4 (ICAM-4) binds to different members of the integrin receptor families. To better define the ICAM-4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that alphaLbeta2, alphaMbeta2, and alphaIIbbeta3 (activated) bind specifically and dose dependently to the recombinant ICAM-4-Fc protein. We also show that cell surface ICAM-4 interacts with the cell surface alphaVbeta3 integrin. In addition, using a alpha4beta1 cell transfectant and beta2 integrin-deficient LAD cells, we show here that ICAM-4 failed to interact with alpha4beta1 even after alpha4beta1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+). ICAM-4 amino acids that are critical for alphaIIbbeta3 and alphaVbeta3 interaction were identified by domain deletion analysis, site-directed mutagenesis and synthetic peptide inhibition. Our results provide evidence that the beta3 integrin binding sites encompass the first and second Ig-like domains of ICAM-4. However, while the alphaIIbbeta3 contact site comprises the ABED face of domain D1 with an extension in the C'-E loop of domain D2, the alphaVbeta3 contact site comprises residues on both faces of D1 and in the C'-E loop of D2. These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM-4, and that ICAM-4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells.     

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

The macrophage alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets

alpha(M)beta(2) integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands. Ligand binding occurs in the alpha(M) chain, which is composed of an I domain and a lectin domain. The alpha(M) I domain binds iC3b, fibrinogen, intercellular adhesion molecule-1, and other ligands and mediates the adhesion of neutrophils to platelet glycoprotein Ibalpha (GPIbalpha). alpha(M)beta(2) also recognizes beta-GlcNAc residues on GPIbalpha that are clustered on platelets after cooling. The phagocytosis of chilled platelets could be reconstituted when Chinese hamster ovary cells were transfected with alpha(M)beta(2). Replacement of the I domain or the lectin domain of the alpha(M) chain with the corresponding domain from the alpha(X) chain (p150) revealed that the activity of the alpha(M)beta(2) integrin toward chilled platelets resides within the lectin domain and does not require the I domain. Additional evidences for this conclusion are: 1) Sf9 cells expressing solely the alpha(M) lectin domain bound chilled platelets, and 2) soluble recombinant alpha(M) lectin domain inhibited the phagocytosis of chilled platelets by alpha(M)beta(2)-expressing THP-1 cells, whereas I domain substrates showed no inhibitory effect. Therefore chilled platelets are removed from blood by an interaction between beta-GlcNAc residues on clustered GPIbalpha and the lectin domain of alpha(M) chain of the alpha(M)beta(2) integrin, distinguishing this interaction from those mediated by the alpha(M) I domain.     

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.