VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin.

We previously showed that fibronectin (FN) synergized with anti-CD3 in induction of CD4+ T cell proliferation, and that VLA-5 acted as a functional FN receptor in a serum-free culture system. In the present study, we showed that VLA-4 is also involved in this CD3-dependent CD4 cell activation through its interaction with the alternatively spliced CS1 domain of FN. When highly purified CD4 cells were cultured on plates coated with anti-CD3 plus synthetic CS1 peptide-IgG conjugate, significant proliferation could be observed. Neither CS1 alone nor anti-CD3 alone induced this activation. This proliferation was completely blocked by anti-VLA beta 1 (4B4) and anti-VLA-4 (8F2), while anti-VLA-5 (monoclonal antibody [mAb] 16 and 2H6) had no effect. These data indicate that VLA-4 mediates CD3-dependent CD4 cell proliferation via the CS1 domain of FN. Anti-VLA-4 also partially (10-40%) inhibited CD4 cell proliferation induced by native FN plus anti-CD3, implying that the CS1 domain is active in the native plasma FN. However, this native FN-dependent proliferation was entirely abolished by addition of anti-VLA-5 alone. Moreover, when native FN-coated plates were pretreated with anti-FN (mAb 333), which blocks RGDS sites but not CS1 sites, no CD4 cell activation could be observed. These results strongly suggest that CD4 cell activation induced by plasma FN/anti-CD3 may be dependent on both VLA4/CS1 and VLA5/RGDS interactions, although the latter interaction may be required for function of the former.

Surface-induced aggregation of type I procollagen.

We have examined the state of aggregation of type I procollagen in the concentration range 5 to 800 micrograms/ml. Electron microscopy typically indicates a high proportion of aggregated material (greater than 50%), when a range of preparative techniques are used. Aggregates of in-register molecules (segment-long-spacing-like aggregates) are frequently observed, often with units of in-register molecules connected via the C-terminal propeptides. In contrast, studies using gel-filtration chromatography and density-gradient ultracentrifugation demonstrate only limited aggregation in solution (less than 5%) even at 800 micrograms/ml. The aggregated material is mainly dimeric and probably not segment-long-spacing-like. We conclude that aggregation of procollagen is strongly favoured by adsorption to a surface when samples are prepared for electron microscopy. The possible relevance of these observations to the fate of procollagen secreted by cells in vivo is discussed.

Morphology of sheet-like assemblies of pN-collagen, pC-collagen and procollagen studied by scanning transmission electron microscopy mass measurements.

At high concentrations, type I pN-collagen, pC-collagen and procollagen (the first 2 generated from procollagen by enzymic cleavage of C-propeptides and N-propeptides, respectively) can all be made to assemble in vitro into thin D-periodic sheets or tapes. Scanning transmission electron microscopy mass measurements show that the pN-collagen sheets and procollagen tapes have a mass per unit area corresponding to that of approximately 6.8 monolayers of close-packed molecules. pN-collagen sheets are extensive and remarkably uniform in mass thickness (fractional S.D. 0.035); procollagen tapes are neither as extensive nor as uniform in thickness. The mean thickness of pC-collagen tapes is less and the variability is greater. In pN-collagen sheets, the overlap: gap mass contrast in a D-period is increased from 5:4 (the ratio in a native collagen fibril) to 6:4, showing that the N-propeptides do not project into the gap but are folded back over the overlap zone. Assuming all N-propeptides to be constrained to the two surfaces of a sheet, their surface density can be found from the mass thickness of the sheet. In a lateral direction (i.e. normal to the axial direction where the spacing is D-periodic), the N-propeptide domains are calculated to be spaced, centre to centre, by 2.23 (+/- 0.1) nm on both surfaces. This value (approx. 1.5 x the triple-helix diameter) implies close-packing laterally with adjacent domains in contact. Sheet formation and the "surface-seeking" behaviour of propeptides can be understood in terms of the dual character of the molecules, evident from solubility data, with propeptides possessing interaction properties very different from those displayed by the rest of the molecule. The form and stability of sheets (and of first-formed fibrils assembling in vivo) could, it is suggested, depend on the partially fluid-like nature of lateral contacts between collagen molecules.

D-periodic assemblies of type I procollagen.

The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide.

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

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.

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

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.

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.

Procollagen type I C-proteinase enhancer is a naturally occurring connective tissue glycoprotein.

Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.

Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression.

Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins alpha 2 beta 1, alpha 5 beta 1, alpha v beta 1 and alpha 6 beta 4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-mediated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-beta 1 antibody, and partial inhibition with anti-alpha 5, suggesting that integrin alpha 5 beta 1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-alpha v and anti-beta 1 antibodies, suggesting that integrin alpha v beta 1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin alpha 6 beta 4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.

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.

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.

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.

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+.

Dynamic aspects of adhesion receptor function--integrins both twist and shout.

The recognition of extracellular molecules by cell surface receptors is the principal mechanism used by cells to sense their environment. Consequently, signals transduced as a result of these interactions make a major contribution to the regulation of cellular phenotype. Historically, particular emphasis has been placed on elucidating the intracellular consequences of growth factor and cytokine binding to cells. In addition to these interactions, however, cells are usually in intimate contact with a further source of complex structural and functional information, namely immobilised extracellular matrix and/or cell surface adhesion proteins. A key question in recent years has been whether cells use the myriad of adhesion protein-receptor interactions purely for structural and migratory function, or whether these interactions also make a more varied contribution to cell phenotype. Here we review dynamic aspects of the function of one major class of adhesion receptor, the integrins. In particular, we focus on the evidence for shape changes in integrin molecules, the mechanisms responsible for regulating ligand binding, and the signals transduced following integrin occupancy.

Mechanisms of integration of cells and extracellular matrices by integrins.

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.

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.