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. 1995 Feb 1;305(Pt 3):945–951. doi: 10.1042/bj3050945

Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29)

T Kamata 1, W Puzon 1, Y Takada 1
PMCID: PMC1136349  PMID: 7531439

Abstract

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arroyo A. G., Sánchez-Mateos P., Campanero M. R., Martín-Padura I., Dejana E., Sánchez-Madrid F. Regulation of the VLA integrin-ligand interactions through the beta 1 subunit. J Cell Biol. 1992 May;117(3):659–670. doi: 10.1083/jcb.117.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aruffo A., Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8573–8577. doi: 10.1073/pnas.84.23.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berlin C., Berg E. L., Briskin M. J., Andrew D. P., Kilshaw P. J., Holzmann B., Weissman I. L., Hamann A., Butcher E. C. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell. 1993 Jul 16;74(1):185–195. doi: 10.1016/0092-8674(93)90305-a. [DOI] [PubMed] [Google Scholar]
  4. Cybulsky M. I., Gimbrone M. A., Jr Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science. 1991 Feb 15;251(4995):788–791. doi: 10.1126/science.1990440. [DOI] [PubMed] [Google Scholar]
  5. D'Souza S. E., Ginsberg M. H., Burke T. A., Lam S. C., Plow E. F. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science. 1988 Oct 7;242(4875):91–93. doi: 10.1126/science.3262922. [DOI] [PubMed] [Google Scholar]
  6. D'Souza S. E., Ginsberg M. H., Burke T. A., Plow E. F. The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit. J Biol Chem. 1990 Feb 25;265(6):3440–3446. [PubMed] [Google Scholar]
  7. Elices M. J., Osborn L., Takada Y., Crouse C., Luhowskyj S., Hemler M. E., Lobb R. R. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. 1990 Feb 23;60(4):577–584. doi: 10.1016/0092-8674(90)90661-w. [DOI] [PubMed] [Google Scholar]
  8. Guan J. L., Hynes R. O. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor alpha 4 beta 1. Cell. 1990 Jan 12;60(1):53–61. doi: 10.1016/0092-8674(90)90715-q. [DOI] [PubMed] [Google Scholar]
  9. Hemler M. E., Huang C., Takada Y., Schwarz L., Strominger J. L., Clabby M. L. Characterization of the cell surface heterodimer VLA-4 and related peptides. J Biol Chem. 1987 Aug 25;262(24):11478–11485. [PubMed] [Google Scholar]
  10. Hemler M. E. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365–400. doi: 10.1146/annurev.iy.08.040190.002053. [DOI] [PubMed] [Google Scholar]
  11. Holzmann B., McIntyre B. W., Weissman I. L. Identification of a murine Peyer's patch--specific lymphocyte homing receptor as an integrin molecule with an alpha chain homologous to human VLA-4 alpha. Cell. 1989 Jan 13;56(1):37–46. doi: 10.1016/0092-8674(89)90981-1. [DOI] [PubMed] [Google Scholar]
  12. Holzmann B., Weissman I. L. Peyer's patch-specific lymphocyte homing receptors consist of a VLA-4-like alpha chain associated with either of two integrin beta chains, one of which is novel. EMBO J. 1989 Jun;8(6):1735–1741. doi: 10.1002/j.1460-2075.1989.tb03566.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu M. C., Crowe D. T., Weissman I. L., Holzmann B. Cloning and expression of mouse integrin beta p(beta 7): a functional role in Peyer's patch-specific lymphocyte homing. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8254–8258. doi: 10.1073/pnas.89.17.8254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kamata T., Puzon W., Takada Y. Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) J Biol Chem. 1994 Apr 1;269(13):9659–9663. [PubMed] [Google Scholar]
  15. Kamata T., Takada Y. Direct binding of collagen to the I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) in a divalent cation-independent manner. J Biol Chem. 1994 Oct 21;269(42):26006–26010. [PubMed] [Google Scholar]
  16. Kina T., Majumdar A. S., Heimfeld S., Kaneshima H., Holzmann B., Katsura Y., Weissman I. L. Identification of a 107-kD glycoprotein that mediates adhesion between stromal cells and hematolymphoid cells. J Exp Med. 1991 Feb 1;173(2):373–381. doi: 10.1084/jem.173.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kovach N. L., Carlos T. M., Yee E., Harlan J. M. A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA-dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components. J Cell Biol. 1992 Jan;116(2):499–509. doi: 10.1083/jcb.116.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Loftus J. C., O'Toole T. E., Plow E. F., Glass A., Frelinger A. L., 3rd, Ginsberg M. H. A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science. 1990 Aug 24;249(4971):915–918. doi: 10.1126/science.2392682. [DOI] [PubMed] [Google Scholar]
  19. Makarem R., Newham P., Askari J. A., Green L. J., Clements J., Edwards M., Humphries M. J., Mould A. P. Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin alpha 4 beta 1. J Biol Chem. 1994 Feb 11;269(6):4005–4011. [PubMed] [Google Scholar]
  20. Masumoto A., Hemler M. E. Multiple activation states of VLA-4. Mechanistic differences between adhesion to CS1/fibronectin and to vascular cell adhesion molecule-1. J Biol Chem. 1993 Jan 5;268(1):228–234. [PubMed] [Google Scholar]
  21. Masumoto A., Hemler M. E. Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin. J Cell Biol. 1993 Oct;123(1):245–253. doi: 10.1083/jcb.123.1.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miyake K., Hasunuma Y., Yagita H., Kimoto M. Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture. J Cell Biol. 1992 Nov;119(3):653–662. doi: 10.1083/jcb.119.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miyake K., Medina K., Ishihara K., Kimoto M., Auerbach R., Kincade P. W. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J Cell Biol. 1991 Aug;114(3):557–565. doi: 10.1083/jcb.114.3.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miyake K., Weissman I. L., Greenberger J. S., Kincade P. W. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med. 1991 Mar 1;173(3):599–607. doi: 10.1084/jem.173.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mould A. P., Komoriya A., Yamada K. M., Humphries M. J. The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues. J Biol Chem. 1991 Feb 25;266(6):3579–3585. [PubMed] [Google Scholar]
  26. Mould A. P., Wheldon L. A., Komoriya A., Wayner E. A., Yamada K. M., Humphries M. J. Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1. J Biol Chem. 1990 Mar 5;265(7):4020–4024. [PubMed] [Google Scholar]
  27. Neuhaus H., Hu M. C., Hemler M. E., Takada Y., Holzmann B., Weissman I. L. Cloning and expression of cDNAs for the alpha subunit of the murine lymphocyte-Peyer's patch adhesion molecule. J Cell Biol. 1991 Nov;115(4):1149–1158. doi: 10.1083/jcb.115.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Osborn L., Hession C., Tizard R., Vassallo C., Luhowskyj S., Chi-Rosso G., Lobb R. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell. 1989 Dec 22;59(6):1203–1211. doi: 10.1016/0092-8674(89)90775-7. [DOI] [PubMed] [Google Scholar]
  29. Prater C. A., Plotkin J., Jaye D., Frazier W. A. The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J Cell Biol. 1991 Mar;112(5):1031–1040. doi: 10.1083/jcb.112.5.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pulido R., Elices M. J., Campanero M. R., Osborn L., Schiffer S., García-Pardo A., Lobb R., Hemler M. E., Sánchez-Madrid F. Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes. J Biol Chem. 1991 Jun 5;266(16):10241–10245. [PubMed] [Google Scholar]
  31. Rice G. E., Bevilacqua M. P. An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science. 1989 Dec 8;246(4935):1303–1306. doi: 10.1126/science.2588007. [DOI] [PubMed] [Google Scholar]
  32. Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. doi: 10.1172/JCI114957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith J. W., Cheresh D. A. Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem. 1990 Feb 5;265(4):2168–2172. [PubMed] [Google Scholar]
  34. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  35. Springer T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994 Jan 28;76(2):301–314. doi: 10.1016/0092-8674(94)90337-9. [DOI] [PubMed] [Google Scholar]
  36. Streeter P. R., Berg E. L., Rouse B. T., Bargatze R. F., Butcher E. C. A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature. 1988 Jan 7;331(6151):41–46. doi: 10.1038/331041a0. [DOI] [PubMed] [Google Scholar]
  37. Sánchez-Madrid F., De Landázuri M. O., Morago G., Cebrián M., Acevedo A., Bernabeu C. VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization. Eur J Immunol. 1986 Nov;16(11):1343–1349. doi: 10.1002/eji.1830161106. [DOI] [PubMed] [Google Scholar]
  38. Takada Y., Elices M. J., Crouse C., Hemler M. E. The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function. EMBO J. 1989 May;8(5):1361–1368. doi: 10.1002/j.1460-2075.1989.tb03516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takada Y., Puzon W. Identification of a regulatory region of integrin beta 1 subunit using activating and inhibiting antibodies. J Biol Chem. 1993 Aug 15;268(23):17597–17601. [PubMed] [Google Scholar]
  40. Takada Y., Ylänne J., Mandelman D., Puzon W., Ginsberg M. H. A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques. J Cell Biol. 1992 Nov;119(4):913–921. doi: 10.1083/jcb.119.4.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Wayner E. A., Kovach N. L. Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin. J Cell Biol. 1992 Jan;116(2):489–497. doi: 10.1083/jcb.116.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamada K. M. Adhesive recognition sequences. J Biol Chem. 1991 Jul 15;266(20):12809–12812. [PubMed] [Google Scholar]
  45. Yednock T. A., Cannon C., Fritz L. C., Sanchez-Madrid F., Steinman L., Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature. 1992 Mar 5;356(6364):63–66. doi: 10.1038/356063a0. [DOI] [PubMed] [Google Scholar]
  46. van de Wiel-van Kemenade E., van Kooyk Y., de Boer A. J., Huijbens R. J., Weder P., van de Kasteele W., Melief C. J., Figdor C. G. Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA. J Cell Biol. 1992 Apr;117(2):461–470. doi: 10.1083/jcb.117.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]

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