Higher order spin resonances in a 2.1-GeV/c polarized proton beam.

Spin resonances can depolarize or spin flip a polarized beam. We studied 1st and higher order spin resonances with stored 2.1 GeV/c vertically polarized protons. The 1st order vertical (ν(y)) resonance caused almost full spin flip, while some higher order ν(y) resonances caused partial depolarization. The 1st order horizontal (ν(x)) resonance caused almost full depolarization, while some higher order ν(x) resonances again caused partial depolarization. Moreover, a 2nd order ν(x) resonance is about as strong as some 3rd order ν(x) resonances, while some 3rd order ν(y) resonances are much stronger than a 2nd order ν(y) resonance. One thought that ν(y) spin resonances are far stronger than ν(x), and that lower order resonances are stronger than higher order; the data do not support this.

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

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.

Molecular basis of ligand recognition by integrin alpha 5beta 1. I. Specificity of ligand binding is determined by amino acid sequences in the second and third NH2-terminal repeats of the alpha subunit.

The NH(2)-terminal portion (putative ligand-binding domain) of alpha subunits contains 7 homologous repeats, the last 3 or 4 of which possess divalent cation binding sequences. These repeats are predicted to form a seven-bladed beta-propeller structure. To map ligand recognition sites on the alpha(5) subunit we have taken the approach of constructing and expressing alpha(V)/alpha(5) chimeras. Although the NH(2)-terminal repeats of alpha(5) and alpha(V) are >50% identical at the amino acid level, alpha(5)beta(1) and alpha(V)beta(1) show marked differences in their ligand binding specificities. Thus: (i) although both integrins recognize the Arg-Gly-Asp (RGD) sequence in fibronectin, the interaction of alpha(5)beta(1) but not of alpha(V)beta(1) with fibronectin is strongly dependent on the "synergy" sequence Pro-His-Ser-Arg-Asn; (ii) alpha(5)beta(1) binds preferentially to RGD peptides in which RGD is followed by Gly-Trp (GW) whereas alpha(V)beta(1) has a broader specificity; (iii) only alpha(5)beta(1) recognizes peptides containing the sequence Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Therefore, amino acid residues involved in ligand recognition by alpha(5)beta(1) can potentially be identified in gain-of-function experiments by their ability to switch the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1). By introducing appropriate restriction enzyme sites, or using site-directed mutagenesis, parts of the NH(2)-terminal repeats of alpha(V) were replaced with the corresponding regions of the alpha(5) subunit. Chimeric subunits were expressed on the surface of Chinese hamster ovary-B2 cells (which lack endogenous alpha(5)) as heterodimers with hamster beta(1). Stable cell lines were generated and tested for their ability to attach to alpha(5)beta(1)-selective ligands. Our results demonstrate that: (a) the first three NH(2)-terminal repeats contain the amino acid sequences that determine ligand binding specificity and the same repeats include the epitopes of function blocking anti-alpha subunit mAbs; (b) the divalent cation-binding sites (in repeats 4-7) do not confer alpha(5)beta(1)- or alpha(V)beta(1)-specific ligand recognition; (c) amino acid residues Ala(107)-Tyr(226) of alpha(5) (corresponding approximately to repeats 2 and 3) are sufficient to change all the ligand binding properties of alpha(V)beta(1) to those of alpha(5)beta(1); (d) swapping a small part of a predicted loop region of alpha(V) with the corresponding region of alpha(5) (Asp(154)-Ala(159)) is sufficient to confer selectivity for RGDGW and the ability to recognize RRETAWA.

Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin.

The interaction of fibronectin with cell surface heparan sulfate proteoglycans is important biologically in inducing reorganization of the cytoskeleton and the assembly of focal adhesions. The major heparan sulfate-binding site in fibronectin, which is also implicated in these morphological events, is the COOH-terminal Hep-2 domain. We describe the first extensive study of the structural determinants required for the interaction between heparan sulfate/heparin and Hep-2. It is clear that, in heparan sulfate, there is a very prominent role for N-sulfate groups, as opposed to a relatively small apparent contribution from carboxyl groups. Furthermore, a minimal octasaccharide binding sequence appeared to contain at least two 2-O-sulfated iduronate residues, but no 6-O-sulfate groups. However, affinity was enhanced by the presence of 6-O-sulfates, and the interaction with Hep-2 also increased progressively with oligosaccharide size up to a maximum length of a tetradecasaccharide. This overall specificity is compatible with recent information on the structure of Hep-2 (Sharma, A., Askari, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) EMBO J. 18, 1468-1479) in which two separate, positively charged clusters, involving up to 11 basic amino acid residues (mostly arginines with their preferential ability to co-ordinate sulfate groups), could form a single extended binding site.

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.

Integrin alpha 4 beta 1-mediated melanoma cell adhesion and migration on vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced IIICS region of fibronectin.

The integrin receptor alpha 4 beta 1 (also known as VLA-4) binds two different ligands, the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1) and the extracellular matrix component fibronectin. Three distinct sites in fibronectin are recognized by alpha 4 beta 1. Two of these (represented by peptides CS1 and CS5) are present in the alternatively spliced IIICS region and lie in separate, independently spliced segments of this region. A third site resides in the adjacent constitutively expressed HepII domain. Recombinant proteins containing the HepII domain and different splice variants of the IIICS have been generated and compared for their ability to mediate cell attachment, spreading and migration. The activity of these proteins has also been compared with that of a recombinant soluble form of VCAM-1 (rsVCAM-1). All the recombinant proteins supported A375-SM human melanoma cell attachment and spreading in an alpha 4 beta 1-dependent manner, but had varied adhesive activities with rsVCAM-1 > fibronectin variants containing the CS1 sequence >> other fibronectin variants. Low concentrations of rsVCAM-1 and CS1-containing fibronectin variants effectively supported cell migration in a trans-filter assay; however, cell motility was retarded at high concentrations of the same proteins. Fibronectin variants lacking CS1 supported little or no migration. To obtain further insight into the molecular basis of this varied adhesive activity, apparent dissociation constants for each of the recombinant proteins were measured using a solid phase receptor-ligand binding assay. The results revealed a hierarchy of ligand affinities that mirrored their adhesive activity (rsVCAM-1 > fibronectin variants containing CS1 >> other fibronectin variants).

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit.

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).

Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin alpha 4 beta 1.

The integrin receptor alpha 4 beta 1 binds to two different ligands, the extracellular matrix glycoprotein fibronectin and the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1). Using probes derived from each ligand and a variety of cell adhesion and ligand-receptor binding assays, we have investigated the relationship between the mechanisms of fibronectin and VCAM-1 interaction with alpha 4 beta 1. CS1 peptide, which represents the dominant active site from the HepII/IIICS recognition domain in fibronectin, was found to inhibit VCAM-1-dependent adhesion in three different assays: MOLT-4 T lymphoblastic leukaemia cell attachment to immobilized recombinant soluble VCAM-1 (rsVCAM-1), MOLT-4 cell attachment to monolayers of VCAM-1-transfected COS-1 cells, and A375-SM melanoma cell spreading on immobilized rs VCAM-1. Half-maximal inhibition required CS1 concentrations of 1.7-3.0 mg/ml, some 3-7-fold higher than that needed to autoinhibit adhesion to CS1-IgG conjugate. Using a more sensitive solid-phase receptor-ligand binding assay, CS1 was found to be a potent inhibitor of the binding of rsVCAM-1 to alpha 4 beta 1 (half-maximal inhibition at 13 micrograms/ml). In agreement with cell-based assays, severalfold lower concentrations of CS1 were required to inhibit binding of recombinant HepII/IIICS region of fibronectin (half-maximal inhibition at 3 micrograms/ml). VCAM-1-alpha 4 beta 1 binding was blocked not only by CS1 peptide but also by the recombinant HepII/IIICS region of fibronectin. Kinetic analysis of CS1 inhibition of VCAM-1 binding revealed that it was directly competitive in nature, indicating that VCAM-1 and fibronectin recognize either identical or spatially overlapping binding sites on alpha 4 beta 1. The implications of these results for the future design of VCAM-1 antagonists are discussed.

Generation of a minimal alpha5beta1 integrin-Fc fragment.

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.

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.

Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

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.