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M J Humphries

Publications and source records attributed to M J Humphries.

At least 55 records · Page 3Linked to original sources
PubMed1998

Fibronectin is a survival factor for differentiated osteoblasts.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.

Animals↗
PubMed1998

Effects of collagenase-cleavage of type I collagen on alpha2beta1 integrin-mediated cell adhesion.

In this paper we show that collagenase-3 cleavage of type I collagen has a marked effect on alpha2beta1 integrin-mediated interactions with the collagen fragments generated. Isolated alpha2beta1 integrin and alpha2 integrin A-domain were found to bind to both native collagen and native 3/4 fragment and, to a lesser degree, native 1/4 fragment. Whole integrin and integrin A-domain binding were lost after heat denaturation of the collagen fragments. At physiological temperature, cell adhesion to triple-helical 3/4 fragment via alpha2beta1 integrin was still possible; however, no alpha2beta1 integrin-mediated adhesion to the 1/4 fragment was observed. Unwinding of the collagen fragment triple helices by heating to physiological temperatures prior to adsorption to plastic tissue culture plates resulted in total abrogation of HT1080 cell attachment to either fragment. These results provide significant evidence in support of a role for matrix-metalloproteinase cleavage of the extracellular matrix in modifying cell-matrix interactions.

Animals↗
PubMed1998

The structure of cell-adhesion molecules.

In recent years, following the identification and molecular cloning of many key adhesion molecules, the three-dimensional structures of some of the domains that mediate adhesive interactions have been determined. This review discusses how these studies have helped explain the unique functional roles of the different families of adhesion molecules.

Cell Adhesion Molecules↗
PubMed1997

Influence of interleukin-3 and other growth factors on alpha4beta1 integrin-mediated adhesion and migration of human hematopoietic progenitor cells.

The mechanisms by which hematopoietic progenitor cells are normally anchored in stromal niches and yet can be mobilized by specific growth factors are poorly understood. It is likely, however, that integrins and their extracellular matrix (ECM) ligands play a key role in this process, and recent evidence suggests that integrin function is modulated by signals originating from activated growth factor receptors. We have now examined this further by studying the role of growth factors on alpha4beta1 integrin-mediated adhesion of human CD34+ hematopoietic progenitor cells to specific recombinant fibronectin fragments coated onto tissue culture dishes. Cells were prepared from cord blood and peripheral blood harvests. During a 30-minute adhesion assay a mean of 74% of CD34 cells attached to the so-called H120 fragment of fibronectin, which contains the strongest alpha4beta1 integrin-binding sequence. The level of cell adhesion was significantly reduced by low concentrations of interleukin-3 (IL-3) (2.5 to 10 ng/mL), whereas stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) at these concentrations did not affect adherence of the cells. Migratory behavior of CD34 cells was examined using fibronectin fragments adsorbed onto a Transwell filter. The H120 fragment supported much higher levels of cell migration than the H0 fragment of fibronectin, which contains a weak alpha4beta1 integrin binding sequence. Over a 16-hour assay, migration of peripheral blood progenitor cells was increased slightly by SCF and by G-CSF. However, a marked stimulation was observed with IL-3, which significantly increased migration. Similar effects were noted with cord blood cells, although a small proportion of cells were able to migrate in the absence of growth factors. These results indicate that there is a highly selective and functional link between the alpha4beta1 integrin and IL-3/IL-3-receptor that could affect the position of stem and progenitor cells in the marrow stroma and influence their growth and development.

Antigens, CD34↗
PubMed1997

An RGD to LDV motif conversion within the disintegrin kistrin generates an integrin antagonist that retains potency but exhibits altered receptor specificity. Evidence for a functional equivalence of acidic integrin-binding motifs.

Integrin ligands almost invariably employ a variant of either the RGD or LDV motif as a key element of their receptor recognition site. These short acidic peptide sequences collaborate with specific nonhomologous flanking residues and spatially separate "synergy" sequences to determine receptor binding specificity. Although the consensus sequences for RGD and LDV motifs are quite different, their common use suggests that they might share a critical role in receptor-ligand engagement. To date, the effects of interconversion of the two motifs within a natural protein framework have not been tested; however, in this study, we have converted the natural RGD site found in the snake venom disintegrin kistrin into an LDV motif and examined the effects of the change on the specificity of integrin recognition and on disintegrin potency. While an assessment of receptor binding using cell adhesion and purified integrin solid-phase assays demonstrated recognition of recombinant RGD kistrin by alphaVbeta3 and alpha5beta1, a series of LDV kistrin chimeras did not bind to these integrins, but instead were recognized specifically by alpha4beta1. The minimal change to elicit this distinct switch in receptor specificity was found to involve alteration of only three residues within kistrin. Alanine scanning mutagenesis was used to provide further information on the functional contribution of the three residues. More important, the LDV kistrin chimeras also retained much of the characteristic potency of RGD kistrin, indicating that the kistrin scaffold is optimized for presentation of both RGD and LDV sequences. These findings provide evidence for similarities in motif pharmacophore and reinforce the hypothesis that RGD and LDV sites have an equivalent functional role in receptor binding. They also demonstrate the potential for other disintegrin-containing proteins, perhaps from the ADAM family, to employ LDV sequences for integrin binding.

Amino Acid Sequence↗
PubMed1997

Alpha4 integrin binding interfaces on VCAM-1 and MAdCAM-1. Integrin binding footprints identify accessory binding sites that play a role in integrin specificity.

Integrins are a family of heterodimeric adhesion receptors that mediate cellular interactions with a range of matrix components and cell surface proteins. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins (alpha4beta1 and alpha4beta7). A related CAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is recognized by alpha4beta7 but is a poor ligand for alpha4beta1. Previous studies have revealed that all alpha4 integrin-ligand interactions are dependent on a key acidic ligand motif centered on the CAM domain 1 C-D loop region. By generating VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell adhesion assays we have found that alpha4beta1 binds to the MAdCAM-1 adhesion motif when present in VCAM-1, but not when the VCAM-1 motif was present in MAdCAM-1, suggesting that this region does not contain all of the information necessary to determine integrin binding specificity. To characterize integrin-CAM specificity further we measured alpha4beta1 and alpha4beta7 binding to a comprehensive set of mutant VCAM-1 constructs containing amino acid substitutions within the predicted integrin adhesion face. These data revealed the presence of key "regulatory residues" adjacent to integrin contact sites and an important difference in the "footprint" of alpha4beta1 and alpha4beta7 that was associated with an accessory binding site located in VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and sequence and when mutated abolishes integrin binding activity.

Amino Acid Sequence↗
PubMed1997

Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.

The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Animals↗
PubMed1997

Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.

The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.

Amino Acid Sequence↗
PubMed1997

Molecular characterisation of integrin-procollagen C-propeptide interactions.

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha2beta1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha2beta1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha2beta1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blocking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha1beta1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha1 and alpha2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin-CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin-C-propeptide interaction does not utilise established peptide motifs.

Amino Acid Sequence↗
PubMed1997

Direct role of the carboxy-terminal cell-binding domain of fibronectin in neural crest cell motility.

We have analyzed the interaction of neural crest cells with fragments of fibronectin corresponding to the different spliced variants of the COOH-terminal cell-binding domain (COOH-ter CBD). We have shown that this domain can support cell adhesion and migration and that both the IIICS and HepII regions are involved in these events. The rate of locomotion is high, although undirectional, compared to that of whole fibronectin. Interactions with the COOH-ter CBD are controlled by alpha4beta1 and maybe other beta1 integrins and cell-surface proteoglycans. These receptors act cooperatively to mediate attachment, spreading, and migration on fibronectin.

Animals↗
PubMed1997

The integrin alpha1 A-domain is a ligand binding site for collagens and laminin.

The integrin alpha1beta1 is a cell surface receptor for collagens and laminin. The alpha1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the alpha1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant alpha1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from alpha1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The alpha2 A-domain, from the alpha2beta1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for alpha1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. alpha1 A-domain bound to all three fragments, while the alpha2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the alpha1beta1 and alpha2beta1 integrins.

Animals↗
PubMed1997

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1997

A structure prediction for the ligand-binding region of the integrin beta subunit: evidence for the presence of a von Willebrand factor A domain.

The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin-ligand binding has been extensively studied because of the potential for the development of anti-adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N-terminus of the beta subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A-domain, thereby potentially placing a previously characterised module at the centre of a key functional region.

Algorithms↗
PubMed1997

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.

Animals↗
PubMed1997

Requirement for Rho in integrin signalling.

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.

3T3 Cells↗
PubMed1996

The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif.

Integrin alpha 2 beta 1 is a major cellular receptor for collagens, but the molecular basis of its function is unknown. The alpha 2 subunit contains a von Willebrand factor A-domain (I-domain) in its N-terminal region, and it has been demonstrated recently that this domain binds specifically to collagen I. This interaction requires divalent cations (e.g., Mg2+) and native collagen conformation, as does binding of the parent integrin to collagen. The alpha 2 A-domain therefore has a number of functional similarities to the parent integrin, alpha 2 beta 1. However, while sequence specificity has been demonstrated for the parent integrin, no such observations have been made for the A-domain. In particular, it is not known whether the A-domain is responsible for sequence-specific recognition of collagens or whether it binds to the genetic collagenous motif. To investigate the ligand specificity of the alpha 2 A-domain, its binding to a range of collagenous ligands has been studied, with cation dependence, collagen triple-helicity, and inhibition by function-blocking antibodies as criteria for specificity. Binding of the parent integrin was examined for comparison. The alpha 2 A-domain was found to bind specifically to collagens I, II, IV and XI. The complement component C1q has a collagenous domain but this was unable to support specific binding of alpha 2 A-domain or alpha 2 beta 1. Furthermore, synthetic triple-helical collagenous peptides failed to act as specific ligands. In conclusion, the alpha 2 A-domain binds specifically to a range of extracellular matrix collagens, but it is not a receptor for all collagenous triple helices. By inference, these findings indicate the existence of an integrin-specific sequence motif within collagenous ligands recognised by the alpha 2 A-domain.

Antigens, CD↗
PubMed1996

The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

Adhesins, Bacterial↗