A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens

Metallothioneins chelate metals and consequently may be a control point of metal homeostasis. Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens. TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue. A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A. thaliana than for T. caerulescens. Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.     

Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells

The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.     

Analysis of the alpha4beta1 integrin-osteopontin interaction

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.     

Canine hepatic lysosomal copper protein: identification as metallothionein

We studied the amino acid sequence of canine hepatic lysosomal copper protein obtained from Bedlington terriers affected by inherited copper toxicosis. The primary structure was determined by manual Edman degradations and carboxypeptidase Y digestions of peptides generated by cleavage of the S-carboxyamidomethylated and S-aminoethylated protein with trypsin. Although the amino terminus was blocked and heterogeneous, the protein showed extensive sequence homology to mammalian metallothioneins. In particular, all cysteinyl residues were conserved, in agreement with their function as metal ligands. The microheterogeneity observed in the amino-terminal part of the molecule indicated the presence of two isoforms in canine liver like those found in most other mammals studied so far.     

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.     

Retinal cyclic-GMP phosphodiesterase gamma-subunit: identification of functional residues in the inhibitory region.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.     

Primary and tertiary structures of the first domain of Panulirus interruptus hemocyanin and comparison of arthropod hemocyanins

The amino acid sequence of the first domain (positions 1-175) of Panulirus interruptus hemocyanin subunit a has been determined. The sequence of residues 1-158 (18-kDa fragment obtained by limited proteolysis) was derived from peptides obtained by digestion of this fragment with CNBr and trypsin and by subdigestion of these peptides with other enzymes. The peptides were sequences automatically or manually. The amino acid sequence has been fitted into the electron-density map at 0.32-nm resolution. The residues of domain 1 are folded into a large, mainly helical, globular part, containing one disulfide bridge, and a smaller part near the molecular twofold axis. The latter part consists of an alpha helix and a beta strand which contains a covalently attached carbohydrate moiety. The sites susceptible to limited proteolytic cleavage of the subunit are discussed. Comparison of the N-terminal sequence with those of other arthropod hemocyanins revealed, besides an N-terminal extension of five residues, the presence of a 21-residue loop (positions 22-42) in the crustacean sequences. This loop contains helix 1.2, a less defined region in the electron-density map. It is absent in chelicerate sequences. Strong evidence is presented that: (a) the structure of the first 21 residues (including helix 1.1) is the same in all arthropod hemocyanins with known amino acid sequence; (b) a stretch containing about 15 residues (including part of helix 1.3) following the 21-residue loop has a different structure in crustaceans and chelicerates; (c) the rest of domain 1 has the same structure again. It is shown that all conserved residues are in the contact region with the other two domains.     

Characterization of the surface topography and putative tertiary structure of the human CD7 molecule

The CD7 gp40 molecule is a member of the Ig gene superfamily and is expressed on T cell precursors before their entry into the thymus during fetal development. N-terminal amino acids 1-107 of CD7 are highly homologous to Ig kappa-L chains whereas the carboxyl-terminal region of the extracellular domain of CD7 is proline-rich and has been postulated to form a stalk from which the Ig domain projects. To define potential functional regions of CD7, we have studied the surface topography of the CD7 Ag by synthesizing peptides corresponding to linear sequences within the CD7 extracellular domains, by raising polyclonal anti-CD7 rabbit sera against these peptides, and by computer analysis of the primary CD7 amino acid sequence. Polyclonal anti-CD7 sera were studied using indirect immunofluorescence, RIA, radioimmunoprecipitation, and Western blot assays. Computer analysis was performed comparing the CD7 sequence with all other known protein sequences. We found that three CD7 epitopes defined by peptides CD7-1A (AA 1-38), CD7-4 (AA 48-74), and CD7-7 (AA 129-146) were available for binding antibody on the surface of the CD7 molecule. Using computer analysis, we transposed the amino acid sequence of the CD7 Ig kappa-like N-terminal domain of CD7 onto the spatial coordinates of REI, a previously reported Ig kappa-molecule highly homologous (48%) to the CD7 N-terminal Ig-like region. Based on computer analysis of this putative CD7 three-dimensional structure, both the CD7-1A and CD7-4 regions protruded from the surface of the N-terminal domain of the CD7 molecule. Finally, comparison of the CD7 transmembrane sequence with CD4 and HIV transmembrane sequences and with respiratory syncytial virus fusion sequences demonstrated similar sequence motifs among these molecules.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.     

Terminal deoxynucleotidyltransferase. Alignment of alpha- and beta-subunits of the core enzyme along the primary translation product.

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.     

Independence of the domains of metallothionein in metal binding

Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To determine if metal binding in one domain affects binding in the other, we prepared peptides corresponding to the regions that enfold the two metal-thiolate clusters. Metal reconstitution studies of these peptides revealed stoichiometries of metal binding similar to those observed within the intact molecule. Thus, the alpha domain coordinates 4 Cd(II), 6 Cu(I), or 6 Ag(I) ions regardless of whether the domain is part of the total protein or is studied as a separate peptide. Likewise, the beta domain binds 3 Cd(II), 6 Cu(I), or 6 Ag(I) ions in both the intact protein and as a separate peptide. If cluster B in intact metallothionein is preformed with Cu(I) or Ag(I), cluster A saturates with either 4 mol eq of Cd(II) or 6 mol eq of Ag(I). Similarly, preformation of the A cluster with Cd(II) does not affect the binding of 6 Cu(I) ions in the B cluster. Therefore, the metal-dependent folding of the protein to create one cluster occurs independent of constraints or influences from the other domain. Formation of the protein with a tetrahedrally coordinated metal in one cluster and a trigonally coordinated metal in the other center is possible.     

Chymotryptic and tryptic peptides of fragment alpha 1-CB3 from bovine corneal collagen. Pinpointing the sites of hexose attachment.

Tryptic peptides of citraconylated fragment alpha1-CB3 and chymotryptic peptides of fragment alpha1-CB3 of bovine corneal collagen were prepared, isolated and characterized. Their amino acid compositions were consistent with the amino acid sequence of fragment alpha1-CB3 from calf skin collagen. Two glycoside sites were identified in bovine corneal fragment alpha1-CB3, one of them being the first located in the overlap region of collagen. The results are related to the uniformly narrow collagen fibres found in cornea and essential for its transparency.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation-dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.