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W S Argraves

Publications and source records attributed to W S Argraves.

At least 73 records · Page 4Linked to original sources
PubMed1990

Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.

Amino Acid Sequence↗
PubMed1990

Merosin, a tissue-specific basement membrane protein, is a laminin-like protein.

Merosin is a basement membrane-associated protein found in placenta, striated muscle, and peripheral nerve. A 3.6-kilobase merosin cDNA clone was isolated from a placental cDNA expression library. The clone contained a 3.4-kilobase open reading frame, the 3' portion of which includes protein sequences of proteolytic fragments of merosin. The deduced amino acid sequence of the merosin polypeptide was similar to that of the COOH-terminal region of the 400-kDa A chain of laminin. This part of laminin forms the large globule at the end of the long arm of the laminin cross and is thought to contain the neurite-promoting site and the major cell binding site(s) in laminin. The sequence identity between merosin and the laminin A chain in this region is nearly 40%. An antiserum against a synthetic peptide from the middle of the merosin cDNA sequence identified a 300-kDa polypeptide in placental extracts, indicating that the merosin polypeptide is similar in size to the laminin A chain. Intact merosin was isolated from placental extracts and shown to be covalently associated with the laminin B chains and to have a cross-like structure similar to that of laminin. The similarities between merosin and laminin show that both proteins are members of the same family of basement membrane proteins.

Amino Acid Sequence↗
PubMed1990

The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.

Animals↗
PubMed1990

Fibulin is an extracellular matrix and plasma glycoprotein with repeated domain structure.

We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by cDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibrillar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 +/- 3 micrograms/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.

Amino Acid Sequence↗
PubMed1989

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin.

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.

Binding Sites↗
PubMed1989

Fibulin, a novel protein that interacts with the fibronectin receptor beta subunit cytoplasmic domain.

A 100 kd protein was isolated from tissue and cell extracts by affinity chromatography on a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit. The 100 kd protein also bound to native fibronectin receptor, and this binding could be reversed with EDTA. Calcium may be the divalent cation required for the binding since the 100 kd protein was found to bind 45Ca2+. The N-terminal amino acid sequence of the 100 kd protein was not similar to any sequence in a protein data base. Immunofluorescent staining of cells cultured on fibronectin showed the 100 kd protein coinciding with the fibronectin receptor beta subunit in sites of substrate contact. Therefore this protein, which we term fibulin, interacts with the fibronectin receptor in vitro and associates with the receptor in vivo. Fibulin is a potential mediator of interactions between adhesion receptors and the cytoskeleton.

Amino Acid Sequence↗
PubMed1989

Structure of the gene for cartilage matrix protein, a modular protein of the extracellular matrix. Exon/intron organization, unusual splice sites, and relation to alpha chains of beta 2 integrins, von Willebrand factor, complement factors B and C2, and epidermal growth factor.

The entire gene for chicken cartilage matrix protein (CMP) has been isolated and characterized by restriction mapping, electron microscopy, nuclease S1 mapping, and sequence analysis. The gene, which is present in a single copy in the chicken genome, is 18 kilobase pairs long and comprises eight exons and seven introns. It has two transcription initiation sites, 8 base pairs from each other. A sequence very homologous to the consensus nuclear factor III binding-site sequence, a CAT- and a TATA-like sequence are found in the promoter region and ATTAAA is used as a polyadenylation signal. The nucleotide sequence defines a primary translation product of 493 amino acids which consists of a 23-amino acid signal peptide and two large repeated domains connected by an epidermal growth factor module. Amino acid sequences homologous to those of the repeated domains are present in the type A repeats of von Willebrand factor, complement factors B and C2, and in the alpha chains of the integrins Mac-1, p150,95, and LFA-1. The exon-intron structure indicates that the CMP gene may have arisen by exon duplication and exon shuffling during evolution. The GT-AG splice rule cannot be applied for the excision of the last intron of the CMP pre-mRNA. The donor splice site of intron G is basically different from the consensus sequence indicating that a novel type of splicing mechanism might exist in cartilage.

Amino Acid Sequence↗
PubMed1988

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides.

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs.

The amino acid sequence of the vitronectin receptor alpha subunit deduced from cDNA is presented. The sequence defines a 1047-amino-acid polypeptide precursor with a putative signal sequence, a large extracellular domain with several sites homologous to calcium binding sites in other proteins, a transmembrane domain, and a 32-amino-acid cytoplasmic domain. The 7-kilobase vitronectin receptor alpha subunit mRNA was found to be expressed in all cell lines examined, including endothelial cells, K562 and HEL leukemia cells, and osteosarcoma cells. In the two leukemia cell lines, the expression of the vitronectin receptor mRNA, as well as that of the fibronectin receptor, was enhanced in the presence of phorbol ester, a treatment known to increase the adhesiveness of these cells. The HEL cells were the only ones among the cell lines tested that also contained the mRNA of the platelet adhesion receptor alpha subunit, glycoprotein IIb. The expression of glycoprotein IIb was slightly enhanced by treatment of the cells with phorbol ester. These results complete the partial cDNA sequence of the vitronectin receptor alpha subunit published previously (Suzuki, S., Argraves, W. S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M. D., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U.S.A., 83, 8614-8618), confirm that the vitronectin receptor, and not IIb, is expressed in endothelial cells, and show that changes in the level of its expression correlate with changes in cell adhesiveness.

Amino Acid Sequence↗
PubMed1987

Chondrocytes from the cartilage proteoglycan-deficient mutant, nanomelia, synthesize greatly reduced levels of the proteoglycan core protein transcript.

Nanomelia is an avian mutation that results in the absence of the major proteoglycan of cartilage. The deficiency in mutant chondrocytes results from their failure to express the cartilage proteoglycan core protein. Using cartilage proteoglycan core protein cDNA probes, the present study shows that nanomelic chondrocytes synthesize greatly reduced levels of this core protein mRNA. The transcript is normal in size and, in addition, comparative analyses of normal and nanomelic genomic DNA show no indication of a major deletion of the core protein gene. These results are interpreted to mean that the mutation affects the regulation of transcription of the cartilage proteoglycan core protein gene.

Aggrecans↗
PubMed1987

Structure of the chicken link protein gene: exons correlate with the protein domains.

The structure of the chicken link protein gene has been determined from a series of genomic clones that cover the entire coding region as well as the complete 3'-untranslated region and a small portion of the 5'-untranslated region. The gene is greater than 80 kilobase pairs long and is present in a single copy in the chicken genome. The link protein gene contains at least five exons with four encoding the entire protein. The domain of link protein that has homologies with immunoglobulin-like proteins and the tandemly repeated hyaluronic acid binding domains are each encoded by separate exons. The exon-intron structure indicates that the link protein gene may have arisen by exon duplication and exon shuffling.

Amino Acid Sequence↗
PubMed1987

Structural features of cartilage matrix protein deduced from cDNA.

cDNAs encoding the Mr 54,000 chicken cartilage matrix protein (CMP) were selected from a cartilage cDNA expression library by immunological means. Antibodies elicited against insert-encoded protein purified from one of the clones reacted specifically with chicken CMP in immunoblots of total cartilage extract, providing positive identification of the cDNA clones isolated. The cDNAs detect a 3.4-kilobase transcript that was present in sternal cartilage and in cartilaginous but not in precartilaginous embryonic limb tissues. The cDNAs code for 416 amino acids of the chicken CMP, including its COOH terminus. There are two striking features in the deduced CMP amino acid sequence: first, it contains a region with significant homologies to repeat sequences in the precursor for epidermal growth factor; and second, it is made up of two large homologous repeat sequences. These results provide the first detailed structural information on the CMP and establish it as a developmentally regulated marker of cartilage differentiation.

Amino Acid Sequence↗
PubMed1987

Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor.

MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of the human fibronectin receptor.

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.

Amino Acid Sequence↗
PubMed1986

cDNA sequences from the alpha subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide.

The fibronectin receptor is a complex of two cell surface glycopeptides that mediate the binding of cells to fibronectin substrata. To study the structure of this receptor, we have isolated cDNA clones coding for the human fibronectin receptor alpha subunit from a lambda gt11 placental cDNA library. The cDNAs code for 229 amino acids from the COOH terminus of the alpha subunit. The deduced sequence has a hydrophobic region with properties characteristic of a membrane-spanning domain. From the membrane-spanning domain to the COOH terminus are 23-28 amino acids that are likely to constitute the cytoplasmic domain. These results establish the fibronectin receptor alpha subunit as an integral membrane protein.

Amino Acid Sequence↗
PubMed1986

Complete amino acid sequence of chicken cartilage link protein deduced from cDNA clones.

cDNA clones coding for chicken cartilage link protein were isolated and sequenced. The DNA sequence for the entire core polypeptide of the mature link protein and the predicted signal peptide consists of 1065 nucleotides. The deduced primary translation product (355 amino acids) has a molecular mass of 40.7 kDa; the calculated molecular mass of the mature link protein core polypeptide (340 amino acids) is 39.06 kDa. The DNA sequence contains two tandemly arranged repeat sequences that may code for repeated functional domains of link protein involved in binding to hyaluronic acid. The mRNAs for chicken link protein are 6.0, 5.8, and 3.0 kilobase pairs, and the difference between the sizes of the RNA species lies in the 3' untranslated region.

Amino Acid Sequence↗
PubMed1986

cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors.

Cells adhere to vitronectin substrates through a cell surface receptor that recognizes an Arg-Gly-Asp sequence in vitronectin. The receptor is a glycoprotein composed of a 150-kDa alpha and a 115-kDa beta subunit. The alpha subunit consists of two disulfide-bonded chains of 125 kDa and 25 kDa. cDNA clones were isolated for the alpha subunit of the vitronectin receptor from a phage lambda gt11 expression library made with RNA from a human fibroblast cell line, IMR-90. The identity of the clones that had been selected from the library based on immunological criteria was verified by comparison of DNA and protein sequences. NH2-terminal sequences were obtained for each of the alpha-subunit chains. The sequence of the 25-kDa chain of the alpha subunit was found in a cDNA clone, and the amino acid sequence deduced from the cDNA establishes the complete amino acid sequence of the 25-kDa chain. This chain contains a membrane-spanning domain as well as a putative intracytoplasmic region that is 32 amino acids long and consists mostly of polar amino acids. Comparison of the cDNA and protein sequences shows that the 25-kDa chain is generated by proteolytic cleavage of an alpha-subunit precursor, the partial sequence of which is contained in the cDNA clones. These clones contain 1910 base pairs of open reading frame and a 3' untranslated sequence. RNA blot hybridization detected one transcript of about 7 kilobases in RNA from fibroblastic and epithelial cells. Together, the cDNA clones cover 4442 bases of this RNA. The alpha-subunit sequence showed strong homology with the sequence of the alpha subunit of fibronectin receptor. Moreover, the NH2-terminal protein sequence of the 125-kDa chain was homologous with the NH2-terminal sequences of two other cell surface proteins, lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen 1 (Mac-1), which have been implicated as receptors for adhesion proteins of leukocytes. These results establish several of the structural features in the vitronectin receptor and suggest the existence of a superfamily of receptors for cell adhesion proteins.

Amino Acid Sequence↗