Abstract
We have identified an integral membrane glycoprotein in rat liver that mediates adhesion of cultured hepatocytes on fibronectin substrata. The protein was isolated by affinity chromatography of detergent extracts on wheat germ lectin-Agarose followed by chromatography of the WGA binding fraction on fibronectin-Sepharose. The glycoprotein (AGp110), eluted at high salt concentrations from the fibronectin column, has a molecular mass of 110 kD and a pI of 4.2. Binding of immobilized AGp110 to soluble rat plasma fibronectin required Ca2+ ions but was not inhibited by RGD peptides. Fab' fragments of immunoglobulins raised in rabbits against AGp110 reversed the spreading of primary hepatocytes attached onto fibronectin-coated substrata, but had no effect on cells spread on type IV collagen or laminin substrata. The effect of the antiserum on cell spreading was reversible. AGp110 was detected by immunofluorescence around the periphery of the ventral surface of substratum attached hepatocytes, and scattered on the dorsal surface. Immunohistochemical evidence and Western blotting of fractionated liver plasma membranes indicated a bile canalicular (apical) localization of AGp110 in the liver parenchyma. Expression of AGp110 is tissue specific: it was found mainly in liver, kidney, pancreas, and small intestine but was not detected in stomach, skeletal muscle, heart, and large intestine. AGp110 could be labeled by lactoperoxidase-catalyzed surface iodination of intact liver cells and, after phase partitioning of liver plasma membranes with the detergent Triton X-114, it was preferentially distributed in the hydrophobic phase. Treatment with glycosidases indicated extensive sialic acid substitution in at least 10 O-linked carbohydrate chains and 1-2 N-linked glycans. Immunological comparisons suggest that AGp110, the integrin fibronectin receptor and dipeptidyl peptidase IV, an enzyme involved in fibronectin-mediated adhesion of hepatocytes on collagen, are distinct proteins.
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- Akiyama S. K., Yamada K. M. Fibronectin. Adv Enzymol Relat Areas Mol Biol. 1987;59:1–57. doi: 10.1002/9780470123058.ch1. [DOI] [PubMed] [Google Scholar]
- Aplin J. D., Hughes R. C., Jaffe C. L., Sharon N. Reversible cross-linking of cellular components of adherent fibroblasts to fibronectin and lectin-coated substrata. Exp Cell Res. 1981 Aug;134(2):488–494. doi: 10.1016/0014-4827(81)90453-5. [DOI] [PubMed] [Google Scholar]
- Becker A., Neumeier R., Park C. S., Gossrau R., Reutter W. Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes. Eur J Cell Biol. 1986 Jan;39(2):417–423. [PubMed] [Google Scholar]
- Ben-Ze'ev A., Robinson G. S., Bucher N. L., Farmer S. R. Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2161–2165. doi: 10.1073/pnas.85.7.2161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bissell D. M., Guzelian P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann N Y Acad Sci. 1980;349:85–98. doi: 10.1111/j.1749-6632.1980.tb29518.x. [DOI] [PubMed] [Google Scholar]
- Bissell D. M., Stamatoglou S. C., Nermut M. V., Hughes R. C. Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading. Eur J Cell Biol. 1986 Mar;40(1):72–78. [PubMed] [Google Scholar]
- Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Burns G. F., Lucas C. M., Krissansen G. W., Werkmeister J. A., Scanlon D. B., Simpson R. J., Vadas M. A. Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells. J Cell Biol. 1988 Sep;107(3):1225–1230. doi: 10.1083/jcb.107.3.1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Butters T. D., Hughes R. C. Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins. Biochem J. 1975 Jul;150(1):59–69. doi: 10.1042/bj1500059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bärmann M., Wadsack J., Frimmer M. A 50 kDa, actin-binding protein in plasma membranes of rat hepatocytes and of rat liver tumors. Biochim Biophys Acta. 1986 Jul 10;859(1):110–116. doi: 10.1016/0005-2736(86)90324-x. [DOI] [PubMed] [Google Scholar]
- Büchsel R., Kreisel W., Fringes B., Hanski C., Reutter W., Gerok W. Localization and turnover of dipeptidylpeptidase IV in the domains of rat liver plasma membrane. Eur J Cell Biol. 1986 Mar;40(1):53–57. [PubMed] [Google Scholar]
- Chadwick C. M., Garrod D. R. Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum. J Cell Sci. 1983 Mar;60:251–266. doi: 10.1242/jcs.60.1.251. [DOI] [PubMed] [Google Scholar]
- Cheresh D. A., Smith J. W., Cooper H. M., Quaranta V. A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells. Cell. 1989 Apr 7;57(1):59–69. doi: 10.1016/0092-8674(89)90172-4. [DOI] [PubMed] [Google Scholar]
- Clegg J. C. Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mixtures using concanavalin A and peroxidase: application to arenavirus and flavivirus proteins. Anal Biochem. 1982 Dec;127(2):389–394. doi: 10.1016/0003-2697(82)90192-0. [DOI] [PubMed] [Google Scholar]
- Dufour S., Duband J. L., Humphries M. J., Obara M., Yamada K. M., Thiery J. P. Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules. EMBO J. 1988 Sep;7(9):2661–2671. doi: 10.1002/j.1460-2075.1988.tb03119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
- Enrich C., Evans W. H., Gahmberg C. G. Fibronectin isoforms in plasma membrane domains of normal and regenerating rat liver. FEBS Lett. 1988 Feb 8;228(1):135–138. doi: 10.1016/0014-5793(88)80602-1. [DOI] [PubMed] [Google Scholar]
- Hahn E., Wick G., Pencev D., Timpl R. Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin. Gut. 1980 Jan;21(1):63–71. doi: 10.1136/gut.21.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanski C., Huhle T., Reutter W. Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen. Biol Chem Hoppe Seyler. 1985 Dec;366(12):1169–1176. doi: 10.1515/bchm3.1985.366.2.1169. [DOI] [PubMed] [Google Scholar]
- Hong W. J., Petell J. K., Swank D., Sanford J., Hixson D. C., Doyle D. Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels. Exp Cell Res. 1989 May;182(1):256–266. doi: 10.1016/0014-4827(89)90296-6. [DOI] [PubMed] [Google Scholar]
- Hosein B., Bianco C. Monocyte receptors for fibronectin characterized by a monoclonal antibody that interferes with receptor activity. J Exp Med. 1985 Jul 1;162(1):157–170. doi: 10.1084/jem.162.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hubbard A. L., Cohn Z. A. The enzymatic iodination of the red cell membrane. J Cell Biol. 1972 Nov;55(2):390–405. doi: 10.1083/jcb.55.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hughes R. C., Stamatoglou S. C. Adhesive interactions and the metabolic activity of hepatocytes. J Cell Sci Suppl. 1987;8:273–291. doi: 10.1242/jcs.1987.supplement_8.15. [DOI] [PubMed] [Google Scholar]
- Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment. J Cell Biol. 1988 Apr;106(4):1289–1297. doi: 10.1083/jcb.106.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphries M. J., Komoriya A., Akiyama S. K., Olden K., Yamada K. M. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J Biol Chem. 1987 May 15;262(14):6886–6892. [PubMed] [Google Scholar]
- Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
- Johansson S., Forsberg E., Lundgren B. Comparison of fibronectin receptors from rat hepatocytes and fibroblasts. J Biol Chem. 1987 Jun 5;262(16):7819–7824. [PubMed] [Google Scholar]
- Johansson S., Gustafson S., Pertoft H. Identification of a fibronectin receptor specific for rat liver endothelial cells. Exp Cell Res. 1987 Oct;172(2):425–431. doi: 10.1016/0014-4827(87)90400-9. [DOI] [PubMed] [Google Scholar]
- Johansson S., Hök M. Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin. J Cell Biol. 1984 Mar;98(3):810–817. doi: 10.1083/jcb.98.3.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johansson S., Kjellén L., Hök M., Timpl R. Substrate adhesion of rat hepatocytes: a comparison of laminin and fibronectin as attachment proteins. J Cell Biol. 1981 Jul;90(1):260–264. doi: 10.1083/jcb.90.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kajiji S., Tamura R. N., Quaranta V. A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 1989 Mar;8(3):673–680. doi: 10.1002/j.1460-2075.1989.tb03425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleinman H. K., Martin G. R., Fishman P. H. Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3367–3371. doi: 10.1073/pnas.76.7.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleinman H. K., McGarvey M. L., Liotta L. A., Robey P. G., Tryggvason K., Martin G. R. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982 Nov 23;21(24):6188–6193. doi: 10.1021/bi00267a025. [DOI] [PubMed] [Google Scholar]
- Kreamer B. L., Staecker J. L., Sawada N., Sattler G. L., Hsia M. T., Pitot H. C. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev Biol. 1986 Apr;22(4):201–211. doi: 10.1007/BF02623304. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Martinez-Hernandez A. The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver. Lab Invest. 1984 Jul;51(1):57–74. [PubMed] [Google Scholar]
- Moreau A., Maurice M., Feldmann G. Analysis of hepatocyte plasma membrane domains during rat development using monoclonal antibodies. J Histochem Cytochem. 1988 Jan;36(1):87–94. doi: 10.1177/36.1.3275714. [DOI] [PubMed] [Google Scholar]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- Ocklind C., Obrink B. Intercellular adhesion of rat hepatocytes. Identification of a cell surface glycoprotein involved in the initial adhesion process. J Biol Chem. 1982 Jun 25;257(12):6788–6795. [PubMed] [Google Scholar]
- Odin P., Obrink B. The cell-surface expression of the cell adhesion molecule cellCAM 105 in rat fetal tissues and regenerating liver. Exp Cell Res. 1988 Nov;179(1):89–103. doi: 10.1016/0014-4827(88)90351-5. [DOI] [PubMed] [Google Scholar]
- Odin P., Tingström A., Obrink B. Chemical characterization of cell-CAM 105, a cell-adhesion molecule isolated from rat liver membranes. Biochem J. 1986 Jun 1;236(2):559–568. doi: 10.1042/bj2360559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plantefaber L. C., Hynes R. O. Changes in integrin receptors on oncogenically transformed cells. Cell. 1989 Jan 27;56(2):281–290. doi: 10.1016/0092-8674(89)90902-1. [DOI] [PubMed] [Google Scholar]
- Rojkind M., Martinez-Palomo A. Increase in type I and type III collagens in human alcoholic liver cirrhosis. Proc Natl Acad Sci U S A. 1976 Feb;73(2):539–543. doi: 10.1073/pnas.73.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375–413. doi: 10.1146/annurev.bi.57.070188.002111. [DOI] [PubMed] [Google Scholar]
- Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
- Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem. 1989 Aug 15;264(23):13369–13372. [PubMed] [Google Scholar]
- Schwarzbauer J. E., Tamkun J. W., Lemischka I. R., Hynes R. O. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983 Dec;35(2 Pt 1):421–431. doi: 10.1016/0092-8674(83)90175-7. [DOI] [PubMed] [Google Scholar]
- Scoazec J. Y., Maurice M., Moreau A., Feldmann G. Analysis of hepatocyte plasma membrane polarity during rat azo dye hepatocarcinogenesis using monoclonal antibodies directed against domain-associated antigens. Cancer Res. 1988 Dec 1;48(23):6882–6890. [PubMed] [Google Scholar]
- Stamatoglou S. C., Hughes R. C., Lindahl U. Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin. J Cell Biol. 1987 Nov;105(5):2417–2425. doi: 10.1083/jcb.105.5.2417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sudhakaran P. R., Stamatoglou S. C., Hughes R. C. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes. Exp Cell Res. 1986 Dec;167(2):505–516. doi: 10.1016/0014-4827(86)90190-4. [DOI] [PubMed] [Google Scholar]
- Tamkun J. W., Hynes R. O. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem. 1983 Apr 10;258(7):4641–4647. [PubMed] [Google Scholar]
- Urushihara H., Yamada K. M. Evidence for involvement of more than one class of glycoprotein in cell interactions with fibronectin. J Cell Physiol. 1986 Mar;126(3):323–332. doi: 10.1002/jcp.1041260302. [DOI] [PubMed] [Google Scholar]
- Wayner E. A., Garcia-Pardo A., Humphries M. J., McDonald J. A., Carter W. G. Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol. 1989 Sep;109(3):1321–1330. doi: 10.1083/jcb.109.3.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wisher M. H., Evans W. H. Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte. Biochem J. 1975 Feb;146(2):375–388. doi: 10.1042/bj1460375. [DOI] [PMC free article] [PubMed] [Google Scholar]