Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells.

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.

Vitronectin in clotting factor IX concentrates.

Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here.

alpha1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins.

Integrins are heterodimeric adhesion receptors consisting of alpha- and beta-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic alpha1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the alpha1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on alpha1beta1-specific substrates. The alpha1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, alpha-actinin and pp125FAK. It seems to account for alpha1-specific signalling, since when peptides that mimic the cytoplasmic domain of alpha1 are transferred into cells, they influence alpha1beta1-specific adhesion, presumably by competing for binding partners. For alpha1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

Overexpression of alpha1beta1 integrin directly affects rat mesangial cell behavior.

BACKGROUND: Glomerular mesangial cell (MC) proliferation, hypertrophy, and abnormal matrix remodeling characterized by increased expression of fibronectin, laminin and collagen type IV, and neoexpression of collagen I and III are the main biological features of progressive glomerulonephritis (GN). Especially, persistent pathological matrix remodeling may lead to glomerular scar formation (glomerular scarring). We reported recently that alpha1beta1 integrin, a major collagen receptor for MCs, may be a potential adhesion molecule for MC-mediated pathological collagen matrix remodeling in GN. METHODS: To address further the direct role of alpha1beta1 integrin in MC behavior, such as cell growth and matrix remodeling, alpha1beta1 integrin was overexpressed in MCs by transfecting an expression vector containing a full-length rat alpha1 integrin cDNA. Flow cytometry and immunoprecipitation analysis were applied for selection of transfectants with a stable expression of the alpha1 integrin subunit. The effect of alpha1beta1 integrin overexpression on MC biology was examined with a 3H-thymidine incorporation assay, flow cytometric analysis of cell size and DNA content, Western blot analysis of a cyclin-dependent-kinase inhibitor, p27Kip1, alpha-smooth muscle actin expression, and a collagen gel contraction assay. RESULTS: The alpha1 transfectants displayed a dramatic inhibition of 3H-thymidine incorporation as compared with the mock transfectants. Increased expression of the alpha1 subunit inversely correlated with cell cycle progression and paralleled the expression of p27Kip1 and alpha-smooth muscle actin, as well as the cell size in MCs. In addition, the alpha1-transfectants were able to enhance collagen matrix reorganization effectively. CONCLUSION: These results indicate that MC-alpha1beta1 integrin expression is a critical determinant of MC phenotypes, including cell growth, cell size, and collagen matrix remodeling ability, and thereby contributes to scar matrix remodeling (sclerosis) in GN.

The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif.

Integrins are heterodimeric transmembrane proteins that mediate substrate adhesion and migration but also the bidirectional transfer of information across the plasma membrane via their cytoplasmic domains. We addressed the question of whether the very short cytoplasmic tail of the alpha1 integrin subunit of alpha1beta1 integrin is required for alpha1beta1-specific adhesion, spreading, and migration. For this purpose we transfected the alpha1 integrin subunit and two cytoplasmically truncated alpha1 subunits into Chinese hamster ovary (CHO) cells. Elimination of the entire cytoplasmic domain of the alpha1 subunit does not affect adhesion but leads to inhibition of spreading and stress fiber formation. The defect in spreading could not be rescued by lysophosphatidic acid, which has been reported to stimulate actin stress fiber formation via Rho. Additionally, deletion of the entire cytoplasmic domain of the alpha1 subunit abolishes migration toward alpha1beta1-specific substrates. Migration and stress fiber formation are similar in CHO-alpha1 cells and CHO cells carrying an alpha1 subunit still containing the conserved GFFKR motif. So, the GFFKR motif of the alpha1 subunit is essential and sufficient for these processes.

Enzymatic quantification of cell-matrix and cell-cell adhesion.

Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of compounds interfering with cell adhesion. Thus, quantification of cell adhesion needs simple and reliable methods that might be applied for both research and diagnostic purposes. This review presents methodological principles of enzymatic approaches for quantification of cell adhesion. In particular, the advantages of exogenous cell labelling with horseradish peroxidase are described.

Effect of von Willebrand Factor and its proteolytic fragments on kinetics of interaction between the light and heavy chains of human factor VIII.

It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (k(on)) for this interaction. The value of the dissociation rate constant (k(off)) for LCh/HCh complex was lower in the presence of Mn2+ (k(off) 4.6x 10(-6) s(-1)) than Ca2+ (k(off) 8.4 x 10(-6) s(-1)) but in both cases vWF had no effect on k(off). This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the k(off) value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1-1365), but not small monomeric N-terminal fragment SPIII-T4 (1-272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of k(on) and did not alter the k(off) value.

Degradation products of factor VIII which can lead to increased immunogenicity.

The biochemical and immunochemical aspects of the development of inhibitors with a plasma-derived, double-virus inactivated factor VIII (FVIII) concentrate (marketed as Octavi SDPlus in Germany and Bisinact in Belgium) are described. A total of 12 cases of inhibitor formation (predominantly type II) were reported in Germany, 8 in Belgium but none in Portugal. Initially, the only difference between the non-pasteurised, SD virus-inactivated product Octavi and the pasteurised product Octavi SDPlus appeared to be pasteurisation, though subsequently, the quality of source material for the product was found to differ in different countries. Separation studies revealed the presence of a 40 kDa peptide fragment in some batches. It was subsequently shown that there was a strong correlation between inhibitor development and batches containing the 40 kDa marker, and a relationship between elevated markers of coagulation activation (FPA in particular) and the occurrence of the 40 kDa marker. Further work revealed that analytical methods commonly used for quality control were not suitable to highlight batch-to-batch differences. It was concluded that inhibitor potential (neoantigenicity) in Octavi SDPlus arose due to two effects; degradation of FVIII already present in source material; and heating of unstable FVIII degradation products. In this case, inhibitors were not caused by the overall production process, nor by GMP failures. The problem of inhibitor potential can be avoided if appropriate preventive measures are taken. Further work is needed to prove non-neoantigenicity and to reinforce the scientific findings, and to characterise pilot batches.

Use of surface plasmon resonance for studies of protein-protein and protein-phospholipid membrane interactions. Application to the binding of factor VIII to von Willebrand factor and to phosphatidylserine-containing membranes.

The surface plasmon resonance phenomenon is used for real time measurements of protein-protein and protein-membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor VIII (fVIII) with von Willebrand factor (vWf) and phospholipid have been developed. These interactions of fVIII are required for maintenance of fVIII concentration in circulation and for the assembly of the functional factor Xase complex, respectively. With these binding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% phosphatidylserine (PS) and 4% PS was examined. The finding that Kd of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), indicates that the heavy chain (HCh) is required for the maximal affinity of fVIII for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles are similar to that of fVIII, indicating that LCh is solely responsible for these interactions. It was also examined how removal of the acidic region affects the binding affinity of the remaining part of LCh for vWf and phospholipid. It was demonstrated that the loss of the LCh acidic region upon thrombin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (k(off) value) and a 165 and 1500-fold increase in the Kd value of the binding of fVIII fragment A3-C1-C2 to vWf compared to that of LCh and fVIII, respectively. In contrast, the binding affinity of A3-C1-C2 for PS-containing membranes was 8-11-fold higher than that of LCh. Possible conformational change(s) in C2 domain upon removal of the acidic region were studied using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3-C1-C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indicates that these conformational changes do occur.

Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB.

Pathologic remodeling of mesangial matrix after glomerular injury is the central biologic feature of glomerular scarring (sclerosis). Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF)-BB have been implicated in the development of glomerular scarring in rat and human glomerulonephritis. To clarify molecular and cellular mechanisms involved in abnormal mesangial remodeling, this study focused on the role of alpha1beta1 integrin, a collagen/laminin receptor, in rat mesangial cells, using collagen gel contraction as an experimental model of in vivo collagen matrix remodeling and scar formation. In addition, the influence of TGF-beta and PDGF-BB on mesangial cell (MC)-mediated collagen gel contraction in association with the alpha1beta1 integrin expression was evaluated. Integrin function blocking studies using anti-alpha1, beta1 subunit antibodies indicated that MC-alpha1beta1 integrin is essentially required not only for collagen-dependent adhesion/migration, but also for gel contraction. Protein synthesis and mRNA analysis experiments demonstrated that TGF-beta, but not PDGF-BB, increases the expression of alpha1beta1 integrin in mesangial cells cultured on plastic surface and in collagen gels. The upregulation of alpha1beta1 integrin expression by TGF-beta correlated with increases in gel contraction and collagen-dependent adhesion but not migration of mesangial cells. On the other hand, PDGF-BB enhanced MC-mediated gel contraction and migration without affecting cell adhesion to collagen I. Growth factor-induced collagen-dependent adhesion, migration, and gel contraction were significantly attenuated by incubation with anti-alpha1, beta1 subunit antibodies. Thus, these data indicate that alpha1beta1 integrin-mediated collagen matrix remodeling can be modulated by TGF-beta and PDGF-BB via different mechanisms. Alpha1 integrin-mediated mesangial matrix remodeling induced by TGF-beta or PDGF-BB may be a pathogenic mechanism leading to glomerular scarring.

Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells.

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.

Rat dipeptidyl peptidase IV (DPP IV) exhibits endopeptidase activity with specificity for denatured fibrillar collagens.

Dipeptidyl peptidase IV (DPP IV, CD 26) is an integral membrane serine protease exhibiting a well characterized exopeptidase activity. The present study shows that DPP IV also possesses a novel gelatinase activity and therefore endopeptidase activity, which was directly demonstrated by gelatin zymography. Protease inhibitor profile analysis showed that the endo- and exopeptidase activities of DPP IV share a common active site. Substrate specificity was detected for denatured collagen types I, II, III and V suggesting that DPP IV might contribute to collagen trimming and metabolism. On the basis of these data we propose that DPP IV and the recently sequenced gelatinolytic seprase (FAPalpha) represent a new subfamily of gelatinolytic integral membrane serine proteases.

Analysis of protein aggregates by combination of cross-linking reactions and chromatographic separations.

Chemical cross-linking provides a method that covalently bridges near-neighbour associations within proteins and protein aggregates. Combined with chromatographic separations and protein-chemical methods, it may be used to localize and to investigate three-dimensional relations as present under natural conditions. This paper reviews the chemistry and application of cross-linking reagents and the development of combination experimental approaches in view of chromatographic separations and cross-linking reactions. Investigations of homooligomeric and heterooligomeric protein associations as well as conformational analysis are presented.

Localization of integrin beta 1, alpha 1, alpha 5 and alpha 9 subunits in the rat testis.

Very late activation (VLA, beta 1; alpha 1; alpha 5, alpha 9) integrins were studied by immunoblotting and immunohistochemistry in the testes of sexually mature rats. All integrin subunits were present in membrane fractions of homogenized testes. Immunohistochemistry revealed that the anti beta 1 antibody recognized peritubular cells and the basement membrane of blood vessels. Immunoreactivity was also demonstrated in the lamina propria, basement membrane, and the basal cytoplasm of Sertoli cells. In elongating spermatids, beta 1 integrin was localized to the acrosome. The alpha 1 subunit was expressed in peritubular cells and in the lamina propria. In the adluminal compartment, round spermatids were stained diffusely for the alpha 1 subunit. Immunoreactivity for alpha 1 integrin was found additionally in the acrosomes of elongating spermatids shortly before their release into the seminiferous tubule lumen. The alpha 5 subunit was expressed in the acrosomes of elongating spermatids as well as in their distal cytoplasm during stages III-VI; the cytoplasmic lobes of elongate spermatids and/or residual bodies also appeared to be immunostained in seminiferous tubules at stages VII-VIII. The alpha 9 subunit was immunolocalized only in the basement membrane and in peritubular cells. These data suggest that integrins are involved in spermatogenesis, in particular in the process of spermatid maturation.

Quantification of cell-matrix and cell-cell adhesion using horseradish peroxidase.

A simple, universal, and rapid enzymatic method for the quantitative determination of cell adhesion in 96-well cell culture plates has been established. The assay is based on cellular steady-state endocytosis, which is used to label cells with horseradish peroxidase (HRP) prior to adhesion. Subsequently, attached cells can be detected by a simple enzymatic reaction, in which the accumulated HRP catalyzes dye formation from a colorless hydrogen donor, e.g., o-phenylenediamine, in the presence of hydrogen peroxide. As demonstrated with different cell lines and test systems, the method can be used to quantify cell-matrix as well as cell-cell interactions and allows a very sensitive quantification of adherent cells. The HRP label is nontoxic and does not affect the adhesion properties of tested cell lines; the quantity of dye formed is proportional to the number of adherent cells. Furthermore, the assay represents an alternative method to isotopic cell labeling, e.g., with 51Cr, which is usually used for quantifying cell-cell interactions.

Cultured dermal papilla cells of the rat vibrissa follicle. Proliferative activity, adhesion properties and reorganization of the extracellular matrix in vitro.

The dermal papilla of the mammalian hair follicle plays an important role in regulating and controlling the hair cycle. Distinct functional stages of dermal papilla cells (DPC) are involved in this process, thus suggesting that the dermal papilla is a highly specialized suborgan of the pilosebaceous unit. The aim of the present study was to investigate the functional properties of cultured DPC in various assays and to compare their functional properties with those of dermal fibroblasts (DFB). In monolayer cell cultures DPC showed an aggregative growth pattern, different to that of DFB, and lower proliferation rates, as compared to the controls. Adhesion assays performed using a 51[Cr]labeling method showed strong adhesion of both cell populations to collagen types I and IV, fibronectin and laminin, but DPC in vitro showed significantly higher adhesiveness to collagen type IV, a major component of the basement membrane of dermal papillae in vivo. The capacity of DPC to reorganize extracellular matrix components, as measured by gel contraction with three-dimensional collagen type I lattices, proved to be significantly lower than that of DFB and, moreover, DPC lysed the collagen lattices completely after 48 h in culture. The functional differences between DPC and DFB were paralleled by higher surface expression and synthesis levels of the beta 1, alpha 1, and alpha 5 chains of integrin adhesion receptors in DPC, as detected by fluorescence-activated cell-sorter analysis and radioimmunoprecipitation. These findings provide evidence that DPC are a highly specialized cell population, which clearly differs from another mesenchymal cell type, DFB. After their isolation and cultivation in vitro, DPC still preserve functional properties related to important steps of cell-matrix interaction involved in the hair cycle.

Chemical cross-linking detects different conformational arrangements of platelet integrin alpha IIb beta III (gpIIb/IIIa).

Treatment of Triton X-100 solubilized platelet membrane with the homobifunctional cross-linker dithiobis(succinimidyl propionate) resulted in covalent cross-linking of the platelet integrin alpha IIb beta 3 (gpIIb/IIIa), the fibrinogen receptor, into three high-molecular-mass complexes with apparent M of 200, 220 and 240 k. Generation of these cross-linked alpha IIb beta 3 aggregates depended on the presence of a native receptor structure and was not influenced by Ca2+ ions and other experimental conditions. Immunoblotting analysis of purified 200/220/240 kDa aggregates revealed that they were made up exclusively by alpha IIb and beta 3 integrin chains in roughly stoichiometric amounts. We therefore conclude that alpha IIb beta 3 integrin occurs in different conformations in the platelet membrane that can be directly detected by chemical cross-linking.

Transforming growth factor-beta (TGF-beta) stimulates the expression of beta1 integrins and adhesion by rat mesangial cells.

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of mesangial matrix (MM) accumulation in human and experimental glomerulonephritis. To clarify molecular mechanisms responsible for pathological MM deposition, we examined the effect of TGF-beta on the production of beta1 integrins and on adhesion function of rat mesangial cells (MC). In immunoprecipitation experiments using [35S]methionine-labeled MC, stimulation of MC with TGF-beta for 48 h resulted in an increase in the synthesis of alpha1beta1 (collagen/laminin receptor) and alpha5beta1 (fibronectin receptor) integrins accompanied by increases in the synthesis of their ligands, collagen type I (collagen I), and fibronectin. A time-dependent increase in beta1, alpha1 integrin subunit mRNA peaking 48 h after exposure to TGF-beta was shown by Northern blot analysis. After 48 h of treatment with TGF-beta, MC displayed significant increases in adhesion to fibronectin, collagen I, and laminin as compared to untreated MC. Anti-beta1 antiserum significantly inhibits MC adhesion to fibronectin, collagen I, and laminin. Anti-alpha1 subunit antibody very strongly inhibited adhesion to collagen I and laminin, but not to fibronectin. Synthetic peptides containing RGD sequences specifically blocked adhesion to fibronectin. These data suggest that TGF-beta may promote MM deposition by increasing MC synthesis of both matrix proteins and beta1 integrins which facilitate binding of these proteins to the MC surface and thus enhance their incorporation into MM.

The cysteine-rich region of dipeptidyl peptidase IV (CD 26) is the collagen-binding site.

A remarkable property of the integral glycoprotein dipeptidyl peptidase IV (DPP IV, CD 26) is its affinity to proteins of the extracellular matrix (ECM). By in vitro binding assays we have shown that DPP IV binds to collagens; preferentially to the collagens I and III, which are both characterized by the formation of large triplehelical domains. No binding of DPP IV to laminin or fibronectin could be observed. Within collagen I, the alpha 1(I) chain was found to be the most prominent binding ligand of DPP IV. A monoclonal anti DPP IV antibody (13.4) specifically inhibited the interaction of DPP IV with collagen I. Peptide mapping and N-terminal sequencing revealed that the corresponding epitope of mAb 13.4 is located in the cysteine-rich domain of DPP IV. We therefore conclude that the putative collagen binding site of DPP IV is different from the region of the catalytic site containing the exopeptidase activity, which is located at the C-terminal portion of the molecule.

Chemical cross-linking leads to two high molecular mass aggregates of rat alpha 1 beta 1 integrin differing in their conformation but not in their composition.

In order to detect protein interactions of the collagen/laminin receptor alpha 1 beta 1 integrin, covalent chemical cross-linking was performed with the homo-bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross-linking of the 190 kDa rat alpha 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M(r) 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of alpha 1 and beta 1 integrin subunits. No other cross-linked proteins associated with the alpha 1 or beta 1 subunit were detected. In contrast to the non-cross-linkable alpha 1 beta 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of alpha 1 beta 1 integrin to cross-link and produce two additional high molecular mass forms is shared by rat alpha 9 beta 1 integrin. Thus, the cross-linking approach directly indicates that beta 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.