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M J Humphries

Publications and source records attributed to M J Humphries.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fine mapping of inhibitory anti-alpha5 monoclonal antibody epitopes that differentially affect integrin-ligand binding.

The high-affinity interaction of integrin alpha5beta1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for alpha5beta1, identified by phage display, which blocks alpha5beta1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-alpha5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-beta1 mAbs, indicating that its epitope is close to the interface between the alpha and beta subunits. Residues in human alpha5 were replaced with the corresponding residues in mouse alpha5 by site-directed mutagenesis; wild-type or mutant human alpha5 was expressed on the surface of alpha5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of alpha5. As expected, disruption of these epitopes had no effect on ligand recognition by alpha5beta1. The locations of these epitopes are consistent with the beta-propeller model for integrin alpha-subunit structure and allow us to propose a topological image of the integrin-ligand complex.

Amino Acid Sequence↗
PubMed1999

Integrin antagonists.

Integrins are a family of cell surface glycoproteins that mediate numerous cell-cell and cell-matrix interactions and are involved in biological processes such as tissue morphogenesis, leukocyte recirculation and migration, wound healing, blood clotting and immune response. Aberrant cell adhesion has been implicated in the pathogenesis of several diseases, including a number of inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and asthma, as well as cancer and coronary heart disease. As such integrins are seen as excellent targets for the development of therapeutic agents. This report begins with an examination of the structure of integrin molecules and their ligands and then goes on to review the current state of development of antiintegrin antagonists.

Animals↗
PubMed1999

Recruitment of a heparan sulfate subunit to the interleukin-1 receptor complex. Regulation by fibronectin attachment.

In this study, we identified an adhesion-regulated subunit of the interleukin-1 (IL-1) receptor complex. Transfection of fibroblasts with an IL-1 receptor-EGFP construct showed that the fusion protein was located at focal adhesions in cells attaching to fibronectin. Fibronectin attachment caused enhancement in endogenous IL-1 type I receptor levels from on average 2500 to 4300 receptors/cell. In addition, matrix attachment resulted in a decrease in binding affinity (Ka) from 1.0 x 10(9) (M-1) to 5.6 x 10(8) (M-1), due to a 2-fold reduction in association rate constant. The adhesion-mediated effects were reversed by soluble heparin. Cross-linking experiments showed that in cells attached to fibronectin, 50-70% of the radiolabeled IL-1 was associated with a heparinase sensitive, high molecular mass component of about 300 kDa, with a core protein of 80-90 kDa. Formation of the complex was dependent on cell interaction with the heparin binding region in fibronectin and required IL-1/type I IL-1 receptor binding. This report demonstrates the recruitment of a heparan sulfate to the IL-1 receptor complex, following attachment to fibronectin, which correlates with alterations in receptor function. The data suggest that the heparan sulfate constitutes an attachment regulated component of the IL-1 receptor complex with the role of mediating matrix regulation of IL-1 responses.

Amino Acid Sequence↗
PubMed1999

PKCalpha regulates beta1 integrin-dependent cell motility through association and control of integrin traffic.

Protein kinase C (PKC) has been implicated in integrin-mediated spreading and migration. In mammary epithelial cells there is a partial co-localization between beta1 integrin and PKCalpha. This reflects complexes between these proteins as demonstrated by fluorescense resonance energy transfer (FRET) monitored by fluorescence lifetime imaging microscopy and also by coprecipitation. Constitutive complexes are observed for the intact PKCalpha and also form with the regulatory domain in an activation-dependent manner. Expression of PKCalpha causes upregulation of beta1 integrin on the cell surface, whereas stimulation of PKC induces internalization of beta1 integrin. The integrin initially traffics to an endosomal compartment in a Ca(2+)/PI 3-kinase/dynamin I-dependent manner and subsequently enters an endocytic recycling pathway. This induction of endocytosis by PKCalpha is a function of activity and is not observed for the regulatory domain. PKCalpha, but not PKCalpha regulatory domain expression stimulates migration on beta1 integrin substrates. This PKCalpha-enhanced migratory response is inhibited by blockade of endocytosis.

Calcium↗
PubMed1999

Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein.

Thrombospondin-related adhesion protein (TRAP) is a Plasmodium falciparum transmembrane protein that is expressed within the micronemes of sporozoites, and is implicated in host cell invasion and motility. Contained within the extracellular region of TRAP is an A-domain, a module found in a number of membrane, plasma and matrix proteins, that is often involved in ligand recognition. In order to determine the role of the TRAP A-domain, it has been expressed as a glutathione S-transferase fusion protein and its ligand binding compared with that of other characterised glutathione S-transferase A-domain fusion proteins. Using a solid phase assay to screen for binding to known A-domain ligands, the TRAP A-domain was found to bind heparin. Binding to heparin appeared to be specific as it was saturable, and was inhibited by soluble heparin, fucoidan and dextran sulfate, but not by other negatively charged sulfated glycosaminoglycans such as chondroitin sulfates. Furthermore, unlike some A-domain ligand interactions, the A-domain of both TRAP and the leukocyte integrin, Mac-1, bound to heparin in the absence of divalent cations. It has been shown previously that another domain within TRAP, which is homologous to region II-plus of circumsporozoite protein, binds to sulfatide and to heparan sulfate on the immortalised hepatocyte line HepG2. The TRAP A-domain also bound to sulfatide and to HepG2 cells. Thus the A-domain shares certain binding properties already attributed to the region II-plus-like domain of TRAP, and may contribute to the binding of TRAP to heparan sulfate on hepatocytes.

Animals↗
PubMed1999

Crystal structure of a heparin- and integrin-binding segment of human fibronectin.

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Amino Acid Sequence↗
PubMed1999

Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.

OBJECTIVE: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production. METHODS: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis. RESULTS: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively. CONCLUSION: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.

Adolescent↗
PubMed1999

Amino acid motifs required for isolated beta cytoplasmic domains to regulate 'in trans' beta1 integrin conformation and function in cell attachment.

The role of beta cytoplasmic domains in regulating beta1 integrin conformation and function in cell attachment is not fully understood. In this study, we tested the ability of transiently expressed beta cytoplasmic domains connected to an extracellular reporter domain to regulate 'in trans' the conformation of endogenous beta1 integrins, and compared these effects on cell attachment. We found that chimeric receptors containing either the beta1, beta3 or beta5 cytoplasmic domains inhibited the expression of the conformationally dependent 9EG7 and 12G10 epitopes on endogenous beta1 integrins. In contrast, chimeric receptors containing the beta4 or alpha5 cytoplasmic domain, or a control receptor lacking a cytoplasmic domain, had no effect. This inhibition occurred in a dose-dependent manner that required high levels of expression of the chimeric receptor. These results suggest that beta1 integrin conformation can be regulated by conserved cytosolic interactions involving beta cytoplasmic domains. This is further supported by our findings that mutations within amino acid motifs conserved among these beta cytoplasmic domains, specifically the NXXY, NPXY and TST-like motifs, reduced the ability of these chimeric receptors to regulate beta1 integrin conformation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST-like motifs. The beta1 chimera also inhibited the binding of soluble fibronectin to endogenous beta1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9EG7 and 12G10 epitopes correlates with integrin function. However, Mn2+, which is an extracellular activator of integrin function, increased 9EG7 expression to basal levels in the presence of the beta1 chimera, but did not rescue cell attachment to the same extent. Thus, although the beta1 integrin conformation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the beta chimeras may be inhibiting both ligand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attachment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function. This finding, together with the requirement for high levels of the chimeric receptors and the fact that mutations in the same conserved motifs in heterodimeric beta1 integrins have been reported to regulate beta1 integrin conformation and function in cell attachment, suggest that beta cytoplasmic domains regulate these processes by interacting with cytosolic factors and that the regulatory effect of the chimeras may be due to their ability to titrate proteins from endogenous integrins.

Amino Acid Sequence↗
PubMed1999

Towards a structural model of an integrin.

Integrins are currently viewed as the principal family of extracellular matrix receptors. The interactions mediated by integrins are responsible for certain typical properties of adhesive cells, such as attachment and migration, but these molecules are also recognized to contribute to intracellular signalling processes, either by transducing signals themselves or by enabling and/or coordinating signalling via other receptor systems. As yet, the structural basis of integrin function is unknown, although detailed computer-based predictions have suggested working models for integrin tertiary structure. In this chapter, I will review this information and discuss recent studies examining the molecular basis of integrin regulation using stimulatory and inhibitory monoclonal antibodies (mAbs). Through the use of sensitive isolated integrin-binding assays, stimulatory mAbs have been found to function either by inducing shape changes in integrins or by selectively recognizing and stabilizing active and ligand-occupied conformations of integrins, while blocking mAbs were found to be allosteric inhibitors of ligand binding that report specific ligand engagement events. This information has improved our understanding of the composition of the integrin ligand-binding pocket and the structural basis of integrin activation.

Antibodies, Monoclonal↗
PubMed1998

Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1.

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha5 beta1, which blocks alpha5 beta1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on alpha5 beta1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha5 and anti-beta1 mAbs to alpha5 beta1 but completely blocked binding of the anti-alpha5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha5 beta1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha5 beta1, and (ii) several mAbs that perturbed binding of alpha5 beta1 to RGD had little effect on binding of alpha5 beta1 to RRETAWA. A double mutation in alpha5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha5 beta1 but had no effect on fibronectin binding or on the binding of other anti-alpha5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha5. Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha5 beta1. Furthermore, as Ser156-Trp157 is unique to the alpha5 subunit, it may be responsible for the specific recognition of RRETAWA by alpha5 beta1.

Amino Acid Sequence↗
PubMed1998

Regulation of the extracellular ligand binding activity of integrins.

Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed.

Animals↗
PubMed1998

Regulation of integrin function: evidence that bivalent-cation-induced conformational changes lead to the unmasking of ligand-binding sites within integrin alpha5 beta1.

The molecular mechanisms that regulate integrin-ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed beta-propeller formed by the N-terminal repeats of the alpha subunit and on the von Willebrand factor A-domain-like region of the beta subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the alpha and beta subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-alpha5 mAbs JBS5 and 16, the inhibitory anti-beta1 mAb 13, and the stimulatory anti-beta1 mAb 12G10 to alpha5 beta1. Analysis of the binding of these mAbs to alpha5beta1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of alpha5 beta1. Competitive ELISA experiments provided evidence that the domains of the alpha5 and beta1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when alpha5 beta1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the alpha subunit beta-propeller and the beta subunit putative A-domain.

Antibodies, Monoclonal↗
PubMed1998

The effect of alpha4 beta1-integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors.

Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between alpha4beta1-integrin-mediated adhesion and growth of CD34(+) cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/IIICS region of fibronectin. CD34(+) cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three alpha4beta1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity alpha4beta1 binding sequences. Proliferation of single cells of CD34(+)38(+)DR+ and CD34(+)38(-)DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P</= .0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P </= .001). The increase in total cell numbers was not at the expense of CD34(+) cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma.

ADP-ribosyl Cyclase↗
PubMed1998

Analysis of ligand-induced and ligand-attenuated epitopes on the leukocyte integrin alpha4beta1: VCAM-1, mucosal addressin cell adhesion molecule-1, and fibronectin induce distinct conformational changes.

The leukocyte integrin alpha4beta1 is a receptor for both cell surface ligands (VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix components (fibronectin). Through regulated interactions with these molecules, alpha4beta1 mediates leukocyte migration from the vasculature at sites of inflammation. Regulation of integrin activity plays a key role in controlling leukocyte-adhesive events and appears to be partly determined by changes in integrin conformation. Several mAbs that recognize ligand-induced binding site epitopes on integrins have been characterized, and a subset of these mAbs are capable of stimulating integrin-ligand binding. Conversely, some mAbs recognize epitopes that are attenuated by ligand engagement and allosterically inhibit ligand binding. To gain insight into ligand-specific effects on integrin conformation, we have examined the ability of different ligands to modulate the binding of four distinct classes (A, B1, B2, and C) of anti-alpha4 Abs to alpha4beta1. VCAM-1 attenuated B (antifunctional) class epitopes via an allosteric mechanism and also allosterically inhibited the binding of the function-blocking anti-beta1 mAb 13. Additional alpha4beta1 ligands (fibronectin fragments, MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding; however, the epitopes of the class B anti-alpha4 mAbs were attenuated by the fibronectin fragments, but not by MAdCAM-1 or the CS1 peptide. Of the two anti-alpha4 class A mAbs examined, one recognized an epitope that was induced uniquely by VCAM-1. Taken together, these data suggest that overlapping but distinct binding mechanisms exist for different alpha4beta1 ligands and that distinct conformational changes are induced upon integrin engagement by different ligands.

Animals↗
PubMed1998

Regulation of macrophage phagocytosis of apoptotic neutrophils by adhesion to fibronectin.

The potential for leukocyte-mediated host tissue damage during resolution of inflammatory responses is influenced by the rate at which extravasated apoptotic leukocytes are cleared from inflammatory sites. Regulation of macrophage capacity for clearance of apoptotic granulocytes is likely to be an important factor determining whether inflammation ultimately resolves or progresses to a chronic state. In this study we have investigated the molecular basis for rapid augmentation of macrophage phagocytosis of apoptotic neutrophils, which was observed following macrophage adhesion to fibronectin. We used a combination of monoclonal antibodies, blocking peptides, and recombinant fibronectin fragments to investigate the role of beta1 integrins in mediating the fibronectin effects. Blockade of alpha5beta1 or alpha4beta1 alone did not attenuate fibronectin-augmentation of phagocytosis. In addition, adhesion of macrophages to recombinant fibronectins lacking alpha4beta1 recognition motifs failed to promote phagocytosis of apoptotic neutrophils. Our results would be consistent with a model in which multiple fibronectin receptors, including beta1 integrins, act co-operatively to augment macrophage phagocytic responses. Together, these data suggest that the extracellular matrix environment of macrophages may provide regulatory signals that act indirectly to rapidly alter the potential for removal of apoptotic cells and influence the process of resolution of inflammation.

Antibodies, Blocking↗
PubMed1998

The integrin beta subunit.

The integrin family of cell adhesion receptors plays a fundamental role in the processes involved in cell division, differentiation and movement. The extracellular domains of integrin alpha/beta heterodimers mediate cell-matrix and cell-cell contacts while their cytoplasmic tails associate with the cytoskeleton. Integrins are capable of transducing information in a bidirectional manner and the beta subunit is now recognised to play an important role in this process. Recent studies have led to the identification of a ligand-binding region on the beta subunit similar to that already characterised on some alpha subunits, and sequences in the cytoplasmic tails of the beta subunits that interact with cytoskeletal and signalling components. Adhesive events can also play a role in the progression of all four major classes of human disease--neoplastic, inflammatory, traumatic and infectious--and the specific nature of integrin adhesion mechanisms make them an attractive target for therapy.

CD18 Antigens↗
PubMed1998

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

Actinin↗