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Arnoud Sonnenberg - One of the best experts on this subject based on the ideXlab platform.
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integrin binding specificity of laminin 10 11 laminin 10 11 are recognized by alpha 3 Beta 1 alpha 6 Beta 1 and alpha 6 Beta 4 integrins
Journal of Cell Science, 2000Co-Authors: Yamato Kikkawa, Noriko Sanzen, Arnoud Sonnenberg, Hironobu Fujiwara, Kiyotoshi SekiguchiAbstract:Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 Beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin Beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 Beta 1 and alpha 6 Beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 Beta 11 and alpha 6 Beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 Beta 1 transfectants. Upon stimulation with the activating anti-integrin Beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and Beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 Beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 Beta 1 and alpha 6 Beta 1 integrins with high avidity and also to alpha 6 Beta 4 integrin.
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mouse egg integrin alpha 6 Beta 1 functions as a sperm receptor
Cell, 1995Co-Authors: Eduardo A C Almeida, Arnoud Sonnenberg, L E Stephens, Ann Sutherland, Aripekka J Huovila, P G Calarco, Leslie M Shaw, Arthur M Mercurio, P Primakoff, D G MylesAbstract:Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, Beta 1, Beta 3, and Beta 5. Immunofluorescence revealed alpha 6 Beta 1 and alpha v Beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v Beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 Beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 Beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 Beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.
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Distinct and overlapping ligand specificities of the alpha 3A Beta 1 and alpha 6A Beta 1 integrins: recognition of laminin isoforms.
Molecular biology of the cell, 1994Co-Authors: G O Delwel, A A De Melker, Frans B L Hogervorst, L H Jaspars, Ingrid Kuikman, D. L. A. Fles, A. Lindblom, Mats Paulsson, R. Timpl, Arnoud SonnenbergAbstract:Abstract The ligand specificity of the alpha 3A Beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A Beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A Beta 1 or alpha 6A Beta 1 integrins. Those expressing alpha 3A Beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and Beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the Beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A Beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A Beta 1 and alpha 6A Beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.
Minoru Fukuda - One of the best experts on this subject based on the ideXlab platform.
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expression cloning of a cdna encoding udp glcnac gal Beta 1 3 galnac r glcnac to galnac Beta 1 6glcnac transferase by gene transfer into cho cells expressing polyoma large tumor antigen
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Marti F A Bierhuizen, Minoru FukudaAbstract:Abstract A cDNA encoding UDP-GlcNAc:Gal Beta 1-3GalNAc-R (GlcNAc to GalNAc) Beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal Beta 1-3(GlcNAc Beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 Beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal Beta 1-3(GlcNAc Beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other Beta 1-6GlcNAc transferases.
James W Dennis - One of the best experts on this subject based on the ideXlab platform.
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increased udp glcnac gal Beta 1 3galnac r glcnac to galnac Beta 1 6 n acetylglucosaminyltransferase activity in metastatic murine tumor cell lines control of polylactosamine synthesis
Journal of Biological Chemistry, 1991Co-Authors: S Yousefi, Elizabeth Higgins, Zhuang Daoling, A Pollexkruger, Ole Hindsgaul, James W DennisAbstract:Abstract Malignant transformation of rodent cell lines by polyoma virus and by activated ras genes is associated with increased UDP-GlcNAc:Man alpha-R Beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-transferase V) activity and it product -GlcNAc Beta 1-6Man alpha 1-6Man Beta 1-branched Asn-linked oligosaccharides. In this report, we have compared Beta 1-6GlcNAc branching of core O- and N-linked oligosaccharides in three experimental models of malignancy, namely (a) rat2 fibroblasts and their malignant T24H-ras-transfected counterpart; (b) benign SP1 mammary carcinoma cells and two metastic sublines of SP1; and (c) the metastatic MDAY-D2 lymphoma cell line and its poorly metastatic glycosylation mutant KBL-1. In addition to the previously reported increase in GlcNAc-transferase V activity, UDP-GlcNAc:Gal Beta 1-3GalNAc alpha-R (GlcNAc to GalNAc) Beta-1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-transferase, EC 2.4.1.102) activity was found to be elevated by 70% in the malignant rat2 and SP1 cell lines while several other glycosyltransferase activities were not significantly different. The action of core 2 GlcNAc-transferase followed by Beta 1-4Gal-transferase provides an N-acetyllactosamine antenna that can be extended with polylactosamine (i.e. repeating Gal Beta 1-4GlcNAc Beta 1-3) provided UDP-GlcNAc:Gal Beta-R Beta 1-3GlcNAc-transferase (GlcNAc-transferase) (i)) activity is present. Polylactosamine content in microsomal membrane glycoproteins was quantitated by labeling the GlcNAc termini resulting from the action of Escherichia freundii endo-Beta-galactosidase with bovine galactosyltransferase/UDP-[3H] Gal. Glycopeptidase F- sensitive and -insensitive fractions were measured to assess the N- and O-linked components. In the SP1 tumor model, the metastatic sublines showed increased core 2 GlcNAc-transferase and GlcNAc-transferase V activities but no change in GlcNAc-transferase (i) activity, yet polylactosamine was increased in both O- and N-linked oligosaccharides. In rat2 cells, down-regulation of GlcNAc-transferase (i) following transformation was associated with decreased polyactosamine even though core 2 GlcNAc-transferase and GlcNAc-transferase V were elevated in the cells. Finally, a 3-fold decrease in GlcNAc-transferase V in KBL-1, the glycosylation mutant of MDAY-D2 cells, resulted in complete loss of polylactosamine in N-linked but no change in O-linked polylactosamine content. These results suggest that, provided GlcNAc-transferase (i) is not limiting, the Beta 1-6-branching enzymes core 2 GlcNAc-transferase and GlcNAc-transferase V regulate the levels of polyactosamine in O- and N-linked oligosaccharides, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Martin J Lohse - One of the best experts on this subject based on the ideXlab platform.
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constitutive activity of the human Beta 1 adrenergic receptor in Beta 1 receptor transgenic mice
Molecular Pharmacology, 2001Co-Authors: Stefan Engelhardt, Yvonne Grimmer, Guohuang Fan, Martin J LohseAbstract:We tested the hypothesis that the human Beta(1)-adrenergic receptor displays constitutive activity and that Beta-adrenergic antagonists differ in their ability to modulate this constitutive activity. Transfection of the cDNAs of the human Beta(1)- and Beta(2)-adrenergic receptors into COS-7 cells caused increases in basal cAMP that were proportional to the receptor levels, thus demonstrating constitutive activity for both subtypes. At comparable receptor levels, the increase in basal cAMP was about 5-fold higher for the Beta(2)- than for the Beta(1)-subtype. As a model for enhanced Beta-adrenergic signaling at the whole-organ level, we used transgenic mice with heart-specific overexpression of the human Beta(1)-adrenergic receptor. In this model, the Beta(1)-adrenergic receptor displayed constitutive activity as evidenced by a higher spontaneous beating rate of isolated right atria from Beta(1)-transgenic versus wild-type mice. This difference was abolished by the addition of CGP20712A, demonstrating inverse agonist properties of this compound. We then tested whether various Beta-adrenergic antagonists currently in clinical use for the treatment of heart failure differ in their ability to modulate constitutive activity of the cardiac Beta(1)-adrenergic receptor. The Beta(1)-selective antagonists metoprolol and bisoprolol showed significant inverse agonist activity at the Beta(1)-adrenergic receptor. Carvedilol behaved as a neutral antagonist and xamoterol displayed marked partial agonist activity. We conclude that the human Beta(1)-adrenergic receptor displays constitutive activity that is considerably lower than that of the Beta(2)-subtype. Beta-Adrenergic antagonists currently in clinical use differ in their ability to exert inverse agonist activity at the human Beta(1)-adrenergic receptor, which may contribute to their therapeutic effects.
Donald Gullberg - One of the best experts on this subject based on the ideXlab platform.
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Beta 1 integrin dependent and independent polymerization of fibronectin
Journal of Cell Biology, 1996Co-Authors: Krister Wennerberg, Martin Pfaff, Donald Gullberg, Lars Lohikangas, Staffan Johansson, Reinhard FasslerAbstract:The mouse cell line GD25, which lacks expression of the Beta 1 family of integrin heterodimers due to disruption of the Beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse Beta 1 integrin subunit. In a stably transformed clone (GD25-Beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-Beta 1A attached to fibronectin and formed focal contacts which contained alpha v Beta 3, but no detectable alpha 5 Beta 1A. The presence of GRGDS peptide allowed alpha 5 Beta 1A to locate to focal contacts of GD25-Beta 1A cultured on fibronectin, while the Beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 Beta 1A and alpha v Beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 Beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v Beta 3 was colocalized with the fibronectin fibrils in GD25-Beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v Beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 Beta 1A and alpha v Beta 3 are able to support adhesion to fibronectin, alpha v Beta 3 dominates in the formation of focal contacts, and alpha 5 Beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of Beta 1 integrins.
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analysis of alpha 1 Beta 1 alpha 2 Beta 1 and alpha 3 Beta 1 integrins in cell collagen interactions identification of conformation dependent alpha 1 Beta 1 binding sites in collagen type i
The EMBO Journal, 1992Co-Authors: Donald Gullberg, K R Gehlsen, D C Turner, Karina Ahlen, L S Zijenah, M J Barnes, Kristofer RubinAbstract:Abstract Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 Beta 1 and alpha 2 Beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 Beta 1, primary rat cardiac fibroblasts alpha 1 Beta 1 and alpha 2 Beta 1, and Rat-1 cells only alpha 2 Beta 1. All three cell types expressed alpha 3 Beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed Beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)
