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. 1998 May 1;331(Pt 3):821–828. doi: 10.1042/bj3310821

Regulation of integrin function: evidence that bivalent-cation-induced conformational changes lead to the unmasking of ligand-binding sites within integrin alpha5 beta1.

A P Mould 1, A N Garratt 1, W Puzon-McLaughlin 1, Y Takada 1, M J Humphries 1
PMCID: PMC1219423  PMID: 9560310

Abstract

The molecular mechanisms that regulate integrin-ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed beta-propeller formed by the N-terminal repeats of the alpha subunit and on the von Willebrand factor A-domain-like region of the beta subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the alpha and beta subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-alpha5 mAbs JBS5 and 16, the inhibitory anti-beta1 mAb 13, and the stimulatory anti-beta1 mAb 12G10 to alpha5 beta1. Analysis of the binding of these mAbs to alpha5beta1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of alpha5 beta1. Competitive ELISA experiments provided evidence that the domains of the alpha5 and beta1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when alpha5 beta1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the alpha subunit beta-propeller and the beta subunit putative A-domain.

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Selected References

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  1. Back A. L., Kwok W. W., Hickstein D. D. Identification of two molecular defects in a child with leukocyte adherence deficiency. J Biol Chem. 1992 Mar 15;267(8):5482–5487. [PubMed] [Google Scholar]
  2. Bazzoni G., Shih D. T., Buck C. A., Hemler M. E. Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J Biol Chem. 1995 Oct 27;270(43):25570–25577. doi: 10.1074/jbc.270.43.25570. [DOI] [PubMed] [Google Scholar]
  3. Corbi A. L., Miller L. J., O'Connor K., Larson R. S., Springer T. A. cDNA cloning and complete primary structure of the alpha subunit of a leukocyte adhesion glycoprotein, p150,95. EMBO J. 1987 Dec 20;6(13):4023–4028. doi: 10.1002/j.1460-2075.1987.tb02746.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. D'Souza S. E., Haas T. A., Piotrowicz R. S., Byers-Ward V., McGrath D. E., Soule H. R., Cierniewski C., Plow E. F., Smith J. W. Ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding. Cell. 1994 Nov 18;79(4):659–667. doi: 10.1016/0092-8674(94)90551-7. [DOI] [PubMed] [Google Scholar]
  5. Huang C., Lu C., Springer T. A. Folding of the conserved domain but not of flanking regions in the integrin beta2 subunit requires association with the alpha subunit. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3156–3161. doi: 10.1073/pnas.94.7.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Huang C., Springer T. A. Folding of the beta-propeller domain of the integrin alphaL subunit is independent of the I domain and dependent on the beta2 subunit. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3162–3167. doi: 10.1073/pnas.94.7.3162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  8. Irie A., Kamata T., Puzon-McLaughlin W., Takada Y. Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits. EMBO J. 1995 Nov 15;14(22):5550–5556. doi: 10.1002/j.1460-2075.1995.tb00242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Irie A., Kamata T., Takada Y. Multiple loop structures critical for ligand binding of the integrin alpha4 subunit in the upper face of the beta-propeller mode 1. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7198–7203. doi: 10.1073/pnas.94.14.7198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kamata T., Puzon W., Takada Y. Identification of putative ligand-binding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29) Biochem J. 1995 Feb 1;305(Pt 3):945–951. doi: 10.1042/bj3050945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B. Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein. Nature. 1994 Jun 23;369(6482):621–628. doi: 10.1038/369621a0. [DOI] [PubMed] [Google Scholar]
  12. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  13. Lanza F., Stierlé A., Fournier D., Morales M., André G., Nurden A. T., Cazenave J. P. A new variant of Glanzmann's thrombasthenia (Strasbourg I). Platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg----214Trp mutation. J Clin Invest. 1992 Jun;89(6):1995–2004. doi: 10.1172/JCI115808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee J. O., Rieu P., Arnaout M. A., Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 1995 Feb 24;80(4):631–638. doi: 10.1016/0092-8674(95)90517-0. [DOI] [PubMed] [Google Scholar]
  15. Loftus J. C., Liddington R. C. Cell adhesion in vascular biology. New insights into integrin-ligand interaction. J Clin Invest. 1997 May 15;99(10):2302–2306. doi: 10.1172/JCI119408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Morris C. A., Underwood P. A., Bean P. A., Sheehan M., Charlesworth J. A. Relative topography of biologically active domains of human vitronectin. Evidence from monoclonal antibody epitope and denaturation studies. J Biol Chem. 1994 Sep 23;269(38):23845–23852. [PubMed] [Google Scholar]
  18. Mould A. P., Akiyama S. K., Humphries M. J. Regulation of integrin alpha 5 beta 1-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+, and Ca2+. J Biol Chem. 1995 Nov 3;270(44):26270–26277. doi: 10.1074/jbc.270.44.26270. [DOI] [PubMed] [Google Scholar]
  19. Mould A. P., Akiyama S. K., Humphries M. J. The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function. J Biol Chem. 1996 Aug 23;271(34):20365–20374. doi: 10.1074/jbc.271.34.20365. [DOI] [PubMed] [Google Scholar]
  20. Mould A. P., Askari J. A., Aota S. i., Yamada K. M., Irie A., Takada Y., Mardon H. J., Humphries M. J. Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit. J Biol Chem. 1997 Jul 11;272(28):17283–17292. doi: 10.1074/jbc.272.28.17283. [DOI] [PubMed] [Google Scholar]
  21. Mould A. P., Garratt A. N., Askari J. A., Akiyama S. K., Humphries M. J. Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the beta 1 subunit. FEBS Lett. 1995 Apr 17;363(1-2):118–122. doi: 10.1016/0014-5793(95)00301-o. [DOI] [PubMed] [Google Scholar]
  22. Mould A. P., Garratt A. N., Askari J. A., Akiyama S. K., Humphries M. J. Regulation of integrin alpha 5 beta 1 function by anti-integrin antibodies and divalent cations. Biochem Soc Trans. 1995 Aug;23(3):395S–395S. doi: 10.1042/bst023395s. [DOI] [PubMed] [Google Scholar]
  23. Mould A. P. Getting integrins into shape: recent insights into how integrin activity is regulated by conformational changes. J Cell Sci. 1996 Nov;109(Pt 11):2613–2618. doi: 10.1242/jcs.109.11.2613. [DOI] [PubMed] [Google Scholar]
  24. Muñoz M., Serrador J., Nieto M., Luque A., Sánchez-Madrid F., Teixidó J. A novel region of the alpha4 integrin subunit with a modulatory role in VLA-4-mediated cell adhesion to fibronectin. Biochem J. 1997 Nov 1;327(Pt 3):727–733. doi: 10.1042/bj3270727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Newham P., Humphries M. J. Integrin adhesion receptors: structure, function and implications for biomedicine. Mol Med Today. 1996 Jul;2(7):304–313. doi: 10.1016/1357-4310(96)10021-6. [DOI] [PubMed] [Google Scholar]
  26. Puzon-McLaughlin W., Takada Y. Critical residues for ligand binding in an I domain-like structure of the integrin beta1 subunit. J Biol Chem. 1996 Aug 23;271(34):20438–20443. doi: 10.1074/jbc.271.34.20438. [DOI] [PubMed] [Google Scholar]
  27. Puzon-McLaughlin W., Yednock T. A., Takada Y. Regulation of conformation and ligand binding function of integrin alpha5beta1 by the beta1 cytoplasmic domain. J Biol Chem. 1996 Jul 12;271(28):16580–16585. doi: 10.1074/jbc.271.28.16580. [DOI] [PubMed] [Google Scholar]
  28. Schiffer S. G., Hemler M. E., Lobb R. R., Tizard R., Osborn L. Molecular mapping of functional antibody binding sites of alpha 4 integrin. J Biol Chem. 1995 Jun 16;270(24):14270–14273. doi: 10.1074/jbc.270.24.14270. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Springer T. A. Folding of the N-terminal, ligand-binding region of integrin alpha-subunits into a beta-propeller domain. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):65–72. doi: 10.1073/pnas.94.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Tozer E. C., Liddington R. C., Sutcliffe M. J., Smeeton A. H., Loftus J. C. Ligand binding to integrin alphaIIbbeta3 is dependent on a MIDAS-like domain in the beta3 subunit. J Biol Chem. 1996 Sep 6;271(36):21978–21984. doi: 10.1074/jbc.271.36.21978. [DOI] [PubMed] [Google Scholar]
  33. Tuckwell D. S., Brass A., Humphries M. J. Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site. Biochem J. 1992 Jul 1;285(Pt 1):325–331. doi: 10.1042/bj2850325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tuckwell D. S., Humphries M. J. A structure prediction for the ligand-binding region of the integrin beta subunit: evidence for the presence of a von Willebrand factor A domain. FEBS Lett. 1997 Jan 6;400(3):297–303. doi: 10.1016/s0014-5793(96)01368-3. [DOI] [PubMed] [Google Scholar]
  35. Wilkins J. A., Li A., Ni H., Stupack D. G., Shen C. Control of beta1 integrin function. Localization of stimulatory epitopes. J Biol Chem. 1996 Feb 9;271(6):3046–3051. [PubMed] [Google Scholar]

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