Biomedical subjects

M J Humphries

Publications and source records attributed to M J Humphries.

At least 73 records · Page 4Linked to original sources

PubMed1996

The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function.

Integrin-ligand binding causes conformational changes in the integrin, as evidenced by the increased expression of epitopes known as ligand-induced binding sites. Some monoclonal antibodies (mAbs) that recognize ligand-induced binding sites stimulate ligand binding, possibly by stabilizing the ligand-occupied conformation of the integrin. Here we have investigated the effect of ligand recognition by alpha5beta1 on the binding of a mAb that inhibits beta1 integrin function (mAb 13). Ligand (fibronectin fragment or GRGDS peptide) decreased the binding of mAb 13 to alpha5beta1. Analysis of this inhibition showed that at high ligand concentrations, approximately 50% of the total integrin bound mAb 13 with >50-fold lower affinity than in the absence of ligand. The concentration of ligand required for half-maximal inhibition of antibody binding was independent of antibody concentration, suggesting that ligand acts as an allosteric inhibitor of mAb 13 binding. Hence, ligand and mAb 13 did not appear to compete directly for binding to alpha5beta1. The stimulatory anti-beta1 mAb 9EG7 was found to increase the maximum level of ligand binding approximately 2-fold, indicating that up to 50% of the total integrin could not bind ligand without 9EG7 stimulation. Analysis of mAb 13 binding in the presence of 9EG7 and ligand (i.e. maximal ligand occupancy) demonstrated that essentially all of the integrin bound mAb 13 with very low or zero affinity. Our results demonstrate that mAb 13 recognizes an epitope that is dramatically attenuated in the ligand-occupied form of alpha5beta1. Hence, since mAb 13 preferentially recognizes the unoccupied conformation of the integrin, the antibody may inhibit ligand binding by stabilizing the unoccupied state of alpha5beta1. In addition, we present evidence that the binding of mAb 13 to ligand-occupied alpha5beta1 may also induce a conformational change in the integrin, resulting in the displacement of ligand.

Allosteric Regulation↗

PubMed1996

Integrin adhesion receptors: structure, function and implications for biomedicine.

Over the past decade, multi-disciplinary approaches have led to the discovery and characterization of several classes of adhesion molecules. Under normal conditions, these molecules provide support for cells, regulate cell migration and contain information that cells use when sensing their environment. In disease, adhesive function is frequently compromised and results in tissue disorder, aberrant cell migration and dysregulation of signalling pathways. The integrins are a major family of adhesion receptors produced by most cell types and are a means by which the cell senses its immediate environment and responds to changes in extracellular matrix composition. Recent years have seen major advances in our understanding of integrin-ligand interactions, and have revealed a structurally dynamic family of receptors capable of translating information into and out of the cell.

Binding Sites↗

PubMed1996

Integrin activation: the link between ligand binding and signal transduction.

A major function of the integrin family of receptors is to provide a physical connection between extracellular adhesion proteins and intracellular cytoskeletal/signalling molecules. These linkages are dynamic and are influenced in a bidirectional manner by changes in the microenvironment of the plasma membrane that occur both inside and outside of cells. The mechanisms employed by integrins to transduce information are complex, but a series of recent studies has clarified their molecular basis. In particular, explanations for the interdependence of ligand binding, occupancy by divalent cations and receptor conformation have been obtained, and some of the key sites responsible for each property have been localized within the integrin heterodimer. These insights now permit a better visualization of the intricate molecular switch that controls the adhesive phenotype.

Animals↗

PubMed1996

Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

Animals↗

PubMed1996

Substratum-dependent stimulation of fibroblast migration by the gelatin-binding domain of fibronectin.

Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or 'Boyden chamber') assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 microgram/ml). The correct assignment of migration-stimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-beta 1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.

Cell Movement↗

PubMed1995

Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+.

Integrin-ligand interactions are known to be dependent on divalent cations, although the precise role of cations in ligand binding is still unclear. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we have performed a comprehensive analysis of the effects of Mn2+, Mg2+, and Ca2+ on ligand binding. Each cation had distinct effects on the ligand-binding capacity of alpha 5 beta 1:Mn2+ promoted high levels of ligand binding, Mg2+ promoted low levels of binding, and Ca2+ failed to support binding. Studies of the effects of different combinations of cations on ligand binding indicated that the cation-binding sites within alpha 5 beta 1 are not all identical, or of broad specificity, but instead each site shows a distinct preference for one or more cations. Ca2+ strongly inhibited Mn(2+)-supported ligand binding, but this inhibition was noncompetitive, suggesting that Ca2+ recognizes different cation-binding sites to Mn2+. In contrast, Ca2+ acted as a direct competitive inhibitor of Mg(2+)-supported ligand binding, implying that Ca2+ can displace Mg2+ from the integrin. However, low concentrations of Ca2+ greatly increased the apparent affinity of Mg2+ for its binding site, suggesting the existence of a distinct high affinity Ca(2+)-binding site. Taken together, our results imply that the ligand-binding capacity of alpha 5 beta 1 can be regulated in a complex manner through separate classes of binding sites for Mn2+, Mg2+, and Ca2+.

Animals↗

PubMed1995

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1.

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

Amino Acid Sequence↗

PubMed1995

Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the beta 1 subunit.

Integrins are the major family of receptors involved in the adhesive interactions of cells with extracellular matrix macromolecules. Although it is known that integrins can exist in active or inactive states, the molecular mechanisms by which integrin activity is modulated are poorly understood. A novel anti-integrin monoclonal antibody, 12G10, that enhances alpha 5 beta 1-fibronectin interactions has been identified. 12G10 binds to the beta 1 subunit and appears to recognise a region of the subunit that contains the epitopes of several previously described activating or inhibitory monoclonal antibodies. However, unlike other activating anti-beta 1 antibodies, the binding of 12G10 to alpha 5 beta 1 is increased in the presence of ligands (fibronectin fragment or RGD peptide). This is the first report for the beta 1 integrin family of an antibody that recognises a ligand-induced binding site, and further emphasises the functional importance of a specific region of the beta 1 subunit in regulating integrin-ligand interactions.

Animals↗

PubMed1995

Effect of beta-mercaptoethanol on the detection of biotinylated proteins.

Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) or conventional autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer onto nitrocellulose. Soaking polyacrylamide gels run under nonreducing conditions in beta-mercaptoethanol (2-ME) prior to protein transfer onto nitrocellulose resulted in a 2- to 10-fold augmentation of the resultant signal. This enhancement was observed for both disulfide- and nondisulfide-bonded proteins. Furthermore, 2-ME had no effect on either the activity of the extravidin-horse-radish peroxidase conjugate, used to detect biotin moieties, or the net protein transfer onto nitrocellulose. Thus, we propose that amplification of either ECL or gamma emission following 2-ME treatment is due to its ability to modify protein conformation, which in turn provides greater access of avidin to biotin.

Biotin↗

PubMed1995

Mechanisms of VCAM-1 and fibronectin binding to integrin alpha 4 beta 1: implications for integrin function and rational drug design.

Integrin alpha 4 beta 1 can mediate both cell-cell and cell-extracellular matrix adhesion by binding to either fibronectin or vascular cell adhesion molecule 1 (VCAM-1). Both interactions are important for extravasation of leukocytes from the blood implying that rationally designed inhibitors of alpha 4 beta 1 function may be useful for treating a various inflammatory conditions. The mechanisms of ligand binding by alpha 4 beta 1 are complicated by the fact that alternative splicing can generate different isoforms of the receptor-binding domains in both fibronectin and VCAM-1. Therefore, in addition to developing alpha 4 beta 1 antagonists, we have also been interested in identifying isoform-specific functions. Recombinant ligand variants have been tested in adhesion and direct receptor-binding assays and each molecule was found to have a different inherent affinity for alpha 4 beta 1 that endows them with different adhesive activities. This suggests that alternative splicing may regulate alpha 4 beta 1-dependent motility in vivo. The initial strategy that we have adopted to develop alpha 4 beta 1 inhibitors has been to identify key amino acid residues and peptide sequences participating in the receptor-ligand binding event and to use this information to generate synthetic mimetics. Three active sites have been identified in fibronectin by testing truncated proteins, expressing recombinant fragments and screening synthetic peptides. Two of these sites employ versions of a novel integrin-binding motif, LDVP/IDAP. A key active site in VCAM-1 has been identified by similar approaches as the related sequence IDSP. Since IDSP-like sequences are probably used by other integrin-binding immunoglobulins, derivatives of these peptides may turn out to be the forerunners of a new generation of therapeutic agents with multiple applications.

Amino Acid Sequence↗

PubMed1995