Structural studies of a neuropeptide precursor protein with an RGD proteolytic site

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.     

Molecular modeling of the thyroid hormone interactions with alpha v beta 3 integrin

A cell surface receptor for thyroid hormone has recently been identified on the extracellular domain of integrin alphavbeta3. In a variety of human and animal cell lines this hormone receptor mediates activation by thyroid hormone of the cellular mitogen-activated protein kinase (MAPK) signal transduction cascade. An arginine-glycine-aspartate (RGD) recognition site on the heterodimeric integrin is essential to the binding of a variety of extracellular matrix proteins. Recent competition data reveal that RGD peptides block hormone-binding by the integrin and consequent MAPK activation, suggesting that the hormone interaction site is located at or near the RGD recognition site on integrin alphavbeta3. A deaminated thyroid hormone (l-thyroxine, T4) analogue, tetraiodothyroacetic acid (tetrac, T4ac), inhibits binding of T4 and 3,5,3'-triiodo-l-thyronine (T3) to alphavbeta3, but does not activate MAPK. Structural data show that the RGD cyclic peptide binds at the interface of the propeller of the alphav and the B domains on the integrin head [Xiong JP, Stehle T, Zhang R, Joachimiack A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alphavbeta3 in complexing with an Arg-Gly-Asp ligand. Science 2002;296:151-5]. To model potential interactions of thyroid hormone analogues with integrin, we mapped T4 and T4ac to the binding site of the RGD peptide. Modeling studies indicate that there is sufficient space in the cavity for the thyroid hormone to bind. Since the hormone is smaller in overall length than the RGD peptide, the hormone does not interact with the Arg recognition site in the propeller domain from alphav. In this model, most of the hormone interactions are with betaA domain of the integrin. Mutagenic studies can be carried out to validate the role of these residues in directing hormone interactions.     

Three-dimensional structure of echistatin and dynamics of the active site

Summary The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization. Disulfide pairings are suggested, based on violations of experimental constraints. The structures satisfy 230 interresidue distance constraints, derived from nuclear Overhauser effect measurements, five hydrogen-bonding constraints, and 21 torsional constraints from vicinal spin-spin coupling constants. The segment from Gly⁵ to Cys²⁰ and from Asp³⁰ to Asn⁴² has a well-defined conformation and the Arg-Gly-Asp sequence, which adopts a turn-like structure, is located at the apex of a nine-residue loop connecting the two strands of a distorted -sheet. The mobility of the Arg-Gly-Asp site has been quantitatively characterized by ¹⁵N relaxation measurements. The overall correlation time of echistatin was determined from fluorescence measurements, and was used in a model-free analysis to determine internal motional parameters. The active site has order parameters of 0.3–0.5, i.e., among the smallest values ever observed at the active site of a protein. Correlation of the flexible region of the protein as characterized by relaxation experiments and the NMR solution structures was made by calculating generalized order parameters from the ensemble of three-dimensional structures. The motion of the RGD site detected experimentally is more extensive than a simple RGD loop wagging motional model, suggested by an examination of superposed solution structures.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Asia1型口蹄疫病毒受体结合位点多样性

【目的】口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)通过结构蛋白VP1 G-H环上高度保守的精氨酸-甘氨酸-天门冬氨酸(Arg-Gly-Asp,RGD)基序与整联蛋白结合起始病毒的感染,但FMDV是RNA病毒,在环境条件变化时,FMDV能够以非RGD的途径起始病毒的感染。为了研究FMDV Asia1/JS/China/05田间舌皮毒经两种不同的途径短期传代后细胞受体结合基序RGD的变异。【方法】采用RT-PCR方法扩增FMDV Asia1/JS/China/05田间毒、田间毒的乳鼠适应毒第四代(MF4)和接种田间毒的牛同居感染的猪水泡病料适应细胞的第八代毒(PBF8)结构蛋白VP1基因,并对不同病毒VP1基因的PCR产物测序和cDNA文库测序。【结果】以含RGD受体结合基序为优势的田间毒在乳鼠上短期传代后出现了含精氨酸-丝氨酸-天门冬氨酸(Arg-Ser-Asp,RSD)和RGD受体结合基序的混合种群,而同居感染后的细胞传代病毒种群则以含精氨酸-天门冬氨酸-天门冬氨酸(Arg-Asp-Asp,RDD)受体结合基序为优势种群。【结论】发现了含RGD受体结合位点为优势的FMDV种群,经不同的宿主短期传代后产了含RSD或RDD受体结合基序的优势种群,该发现不仅增加了保守基序RGD发生替换的FMDV变异株的数量,而且为FMDV的细胞识别和宿主嗜性的改变等进一步研究奠定了物质基础。     

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.     

Non-RGD domains of osteopontin promote cell adhesion without involving αv integrins

Osteopontin (OPN) is an integrin-binding secreted protein that contains an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell types via RGD-mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N-terminal or C-terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16-BL6 cells was not inhibited by either GRGDS peptides or the anti-αv antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C-terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti-αv antibody. Together these results indicate that B16-BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN. © 1996 Wiley-Liss, Inc.     

含RGD受体结合位点口蹄疫病毒Asia1/JS/China/2005株的拯救及初步鉴定

摘要：【目的】构建含有RGD受体结合位点口蹄疫病毒（FMDV）Asia1/JS/China/2005株的全长感染性cDNA克隆。【方法】采用定点突变方法,构建Asia1型FMDV含有预期突变的全长cDNA克隆pFMDV-RGD。pFMDV-RGD重组质粒经NotI线化后,与表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,进行FMDV-RGD病毒拯救。【结果】序列测定结果表明成功构建了FMDV含有RGD受体位点的Asia1/JS/China/2005全长cDNA克隆。共转染实验获得拯救病毒,对拯救的病毒分别进行序列测定、间接免疫荧光、电子显微镜观察和乳鼠致病性分析,表明成功拯救了含有RGD受体结合位点的Asia1/JS/China/2005株FMDV。【结论】该实验为进一步研究含有RGD和RDD受体结合位点2个拯救病毒生物学特性的差异奠定了基础。     

Sequence requirements for prohormone processing in mouse pituitary AtT-20 cells. Analysis using prorenins as model substrates

Although cleavage of peptides at sites marked by paired basic amino acids is a common feature of prohormone processing, little is known about the properties of endoprotease(s) responsible for cleavage of the precursor. To examine the cleavage specificity of a processing endoprotease, we have altered the Lys-Arg cleavage site of human prorenin to Arg-Arg, Lys-Lys and Arg-Lys by site-directed mutagenesis, and expressed the native and mutated precursors in mouse pituitary AtT-20 cells which are known to process foreign prohormones, including prorenin, at paired basic sites during the regulated secretory process. All native and mutated human prorenins were sorted into the regulated secretory pathway. The mutated precursor with Arg-Arg instead of the Lys-Arg native pair was processed at about half the efficiency of the native one, while the Lys-Lys and Arg-Lys mutants were not processed. Rat prorenin, which naturally has a Lys-Lys pair, was not processed in the cells. In addition, mouse Ren2 prorenin, which has a Ser residue next to the Lys-Arg pair, but not mouse Ren1 prorenin, which has a Pro residue next to the pair, was processed. These results suggest that the Arg residue at the COOH side of the basic pair is essential for cleavage of prorenins by a processing enzyme during the regulated secretory process in AtT-20 cells, although the NH2-side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg-Pro peptide bond.     

Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: Analysis by site-directed mutagenesis

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and >200-fold, respectively. The conservative D51E mutant was still >100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa. © 1993 Wiley-Liss, Inc.     

Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning

Two different insulin-like growth-factor (IGF)-binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF-binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N-terminal and C-terminal clusters and an Arg-Gly-Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein-sequence polymorphism was detected at position Ile/Met-228, indicating possible allelic variation. The 3'-untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence, which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth-regulating proteins.     

重组七鳃鳗富含半胱氨酸RGD蛋白rLj-RGD1对人肝癌HepG2细胞的抑制作用

rLj-RGD1为源于日本七鳃鳗口腔腺的基因重组蛋白,其富含半胱氨酸并具有一个RGD(Arg-Gly-Asp)模体.前期工作表明,rLj-RGD1具有抑制血小板聚集及血管新生的RGD毒素蛋白典型功能.为了研究rLj RGD1是否具有RGD毒素蛋白的另一典型抗肿瘤功能,以人肝癌HepG2细胞为模型,对rLj RGD1进行了活性研究.MTT法结果显示,rLj-RGD1呈剂量依赖方式抑制HepG2细胞的增殖,其半抑制浓度(IC50)为36 μmol/L;细胞迁移与浸润实验结果显示,rLj-RGD1能以剂量依赖方式抑制HepG2细胞碱性成纤维生长因子（bFGF）诱导的迁移与浸润.Hoechst染色和DNA Ladder等细胞凋亡实验表明,rLj-RGD1能够以剂量依赖方式诱导HepG2细胞发生凋亡.细胞黏附实验表明,rLj-RGD1以剂量依赖方式抑制HepG2细胞与玻连蛋白(vitronectin, VN)的黏附.上述结果表明,rLj-RGD1具有抑制人肝癌HepG2细胞增殖、迁移和浸润的功能,并可诱导其发生失巢凋亡. 本研究结果提示,rLj-RGD1具有典型的RGD毒素蛋白抗肿瘤功能,其未来具有成为抗肿瘤药物的潜力.     

Molecular properties of poly(RGD) and its binding capacities to metastatic melanoma cells.

We examined the binding capacity of anti-metastatic polypeptide containing repetitive Arg-Gly-Asp(RGD) sequence derived from cell binding site of fibronectin, poly(RGD), to the surface of tumor cells. Poly(RGD) competitively inhibited the binding of radiolabeled fibronectin to the cell surface more potently than oligo(RGD) or RGD tripeptide on a molar basis. Compared on a weight basis to oligo(RGD) or RGD peptide, poly(RGD) was more active than the oligo- and monomeric peptide at inhibiting tumor cell adhesion to immobilized fibronectin. The secondary structure of poly(RGD) was predicted to be a beta-turn from the data of CD spectra and its amino acid sequence. These findings suggest that poly(RGD)-mediated inhibition of cell adhesion is due to its potent binding capacity to fibronectin receptors on cell surface probably through its conformational properties.     

Two enzymes concerned in peptide hormone alpha-amidation are synthesized from a single mRNA.

By expressing truncated rat pituitary 'peptidylglycine alpha-amidating enzyme' cDNAs in COS-7 cells, we found that the two reactions concerned in peptide carboxyl-terminal amidation, namely the peptidylglycine alpha-hydroxylation reaction and the peptidyl-hydroxyglycine amidation reaction, were catalyzed by 37- and 53-K proteins, which were derived from the 5'- and 3'-coding sequences, respectively. The full-length cDNA directed the expression of both the 37- and 53-K enzymes, and in the combined presence of the two enzymes the full conversion of a glycine-extended peptide into the amidated product was achieved. These results indicated that two enzymes concerned in peptide hormone alpha-amidation are generated from a common precursor protein encoded by a single mRNA.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.     

The cyclized C-terminal dipeptide of arginine vasopressin: Metabolic stability and antagonism of puromycin-induced amnesia

The cyclized derivative of the C-terminal dipeptide of arginine vasopressin (AVP), cyclo(Arg-Gly), can, when administered peripherally, block puromycininduced amnesia in mice. The potency and time course of action of this peptide are very similar to that of the parent hormone, AVP. Radioactively labelled cyclo(Arg-Gly), injected sc, was found to be degraded mainly by kidney and liver, and both intact peptide and degradation products reached the brain. However, cyclo(Arg-Gly) injected directly into brain was stable for up to 24 hr. The disappearance of cyclo(Arg-Gly) from plasma and brain was biphasic, with halflives longer than that reported for AVP. The results suggest that cyclo(Arg-Gly), formed in vivo from AVP, could contribute to the long-term effects of AVP in the CNS. However, not all of the actions of the hormone can be attributed to formation of longer-acting derivatives, and it is postulated that separate receptors may exist in brain for the neurohypophyseal hormones and their active fragments.     

Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides.

The Arg-Gly-Asp (RGD) sequence is a universal cell-recognition site of various extracellular proteins that interact with integrin cell-surface receptors. In order to design low-molecular-mass RGD protein antagonists, the determination of the biologically active conformation is a prerequisite. We present a method that yields detailed insight into the steric factors which govern the binding of the ligands to their receptors by systematically scanning the conformational space accessible for the tripeptide sequence RGD. The investigation is based on the conformationally controlled design of homodetic cyclic oligopeptides and their structural determination, coupled with biological assays. For this purpose, a whole set of cyclic pentapeptides and hexapeptides has been synthesized and their three-dimensional structures in solution analyzed by modern two-dimensional NMR techniques in combination with restrained and free molecular dynamics simulations. Their biological activity was compared with that of linear GRGDS in inhibition assays of tumor cell adhesion to laminin P1 and vitronectin substrates. An up to 100-fold, and in part selective, increase in activity was observed for two cyclic pentapeptides. Most other peptides showed a decreased activity which, however, was useful to correlate activity with rather small variations in conformation. Detailed comparative studies of the systematically designed conformations and the corresponding anti-adhesive activities offer an access to lead structures for a rational indirect drug design of peptide and peptidomimetic pharmaceuticals with strong interfering activity for integrin-mediated cell-cell and cell-matrix interactions.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.