Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jan 15;377(Pt 2):449–457. doi: 10.1042/BJ20030411

A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin.

Suresh K Alahari 1, Hani Nasrallah 1
PMCID: PMC1223876  PMID: 14535848

Abstract

In a previous study [Alahari, Lee and Juliano (2000) J. Cell Biol. 151, 1141-1154], we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the alpha5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin-alpha5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and alpha5 were created. The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction. Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the alpha5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and alpha5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected. Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized alpha5 peptides. In addition, mutational analysis revealed that residues Tyr(1018) and Lys(1022) are crucial for alpha5-nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the alpha5 cytoplasmic domain. Further studies demonstrated that the minimal alpha5 binding region of nischarin does not affect cell migration.

Full Text

The Full Text of this article is available as a PDF (199.7 KB).

Selected References

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

  1. Alahari S. K., Lee J. W., Juliano R. L. Nischarin, a novel protein that interacts with the integrin alpha5 subunit and inhibits cell migration. J Cell Biol. 2000 Dec 11;151(6):1141–1154. doi: 10.1083/jcb.151.6.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alahari Suresh K. Nischarin inhibits Rac induced migration and invasion of epithelial cells by affecting signaling cascades involving PAK. Exp Cell Res. 2003 Aug 15;288(2):415–424. doi: 10.1016/s0014-4827(03)00233-7. [DOI] [PubMed] [Google Scholar]
  3. Armulik A., Nilsson I., von Heijne G., Johansson S. Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J Biol Chem. 1999 Dec 24;274(52):37030–37034. doi: 10.1074/jbc.274.52.37030. [DOI] [PubMed] [Google Scholar]
  4. Barry William T., Boudignon-Proudhon Christel, Shock David D., McFadden Andrew, Weiss Jonathan M., Sondek John, Parise Leslie V. Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain. J Biol Chem. 2002 May 21;277(32):28877–28883. doi: 10.1074/jbc.M202983200. [DOI] [PubMed] [Google Scholar]
  5. Bauer J. S., Varner J., Schreiner C., Kornberg L., Nicholas R., Juliano R. L. Functional role of the cytoplasmic domain of the integrin alpha 5 subunit. J Cell Biol. 1993 Jul;122(1):209–221. doi: 10.1083/jcb.122.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berrier A. L., Mastrangelo A. M., Downward J., Ginsberg M., LaFlamme S. E. Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains. J Cell Biol. 2000 Dec 25;151(7):1549–1560. doi: 10.1083/jcb.151.7.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calderwood David A., Yan Boxu, de Pereda Jose M., Alvarez Begoña García, Fujioka Yosuke, Liddington Robert C., Ginsberg Mark H. The phosphotyrosine binding-like domain of talin activates integrins. J Biol Chem. 2002 Apr 3;277(24):21749–21758. doi: 10.1074/jbc.M111996200. [DOI] [PubMed] [Google Scholar]
  8. Carrick F. E., Forbes B. E., Wallace J. C. BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in both the amino- and carboxyl-terminal domains. J Biol Chem. 2001 May 16;276(29):27120–27128. doi: 10.1074/jbc.M101317200. [DOI] [PubMed] [Google Scholar]
  9. Chang D. D., Wong C., Smith H., Liu J. ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin. J Cell Biol. 1997 Sep 8;138(5):1149–1157. doi: 10.1083/jcb.138.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang David D., Hoang Bao Q., Liu Jenny, Springer Timothy A. Molecular basis for interaction between Icap1 alpha PTB domain and beta 1 integrin. J Biol Chem. 2001 Dec 7;277(10):8140–8145. doi: 10.1074/jbc.M109031200. [DOI] [PubMed] [Google Scholar]
  11. Garcia-Mira Maria M., Sadqi Mourad, Fischer Niels, Sanchez-Ruiz Jose M., Muñoz Victor. Experimental identification of downhill protein folding. Science. 2002 Dec 13;298(5601):2191–2195. doi: 10.1126/science.1077809. [DOI] [PubMed] [Google Scholar]
  12. García-Alvarez Begoña, de Pereda José M., Calderwood David A., Ulmer Tobias S., Critchley David, Campbell Iain D., Ginsberg Mark H., Liddington Robert C. Structural determinants of integrin recognition by talin. Mol Cell. 2003 Jan;11(1):49–58. doi: 10.1016/s1097-2765(02)00823-7. [DOI] [PubMed] [Google Scholar]
  13. Geiger C., Nagel W., Boehm T., van Kooyk Y., Figdor C. G., Kremmer E., Hogg N., Zeitlmann L., Dierks H., Weber K. S. Cytohesin-1 regulates beta-2 integrin-mediated adhesion through both ARF-GEF function and interaction with LFA-1. EMBO J. 2000 Jun 1;19(11):2525–2536. doi: 10.1093/emboj/19.11.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Giancotti F. G., Ruoslahti E. Integrin signaling. Science. 1999 Aug 13;285(5430):1028–1032. doi: 10.1126/science.285.5430.1028. [DOI] [PubMed] [Google Scholar]
  15. Hannigan G. E., Leung-Hagesteijn C., Fitz-Gibbon L., Coppolino M. G., Radeva G., Filmus J., Bell J. C., Dedhar S. Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature. 1996 Jan 4;379(6560):91–96. doi: 10.1038/379091a0. [DOI] [PubMed] [Google Scholar]
  16. Hornby Judith A. T., Codreanu Simona G., Armstrong Richard N., Dirr Heini W. Molecular recognition at the dimer interface of a class mu glutathione transferase: role of a hydrophobic interaction motif in dimer stability and protein function. Biochemistry. 2002 Dec 3;41(48):14238–14247. doi: 10.1021/bi020548d. [DOI] [PubMed] [Google Scholar]
  17. Hughes P. E., Diaz-Gonzalez F., Leong L., Wu C., McDonald J. A., Shattil S. J., Ginsberg M. H. Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem. 1996 Mar 22;271(12):6571–6574. doi: 10.1074/jbc.271.12.6571. [DOI] [PubMed] [Google Scholar]
  18. Hughes P. E., O'Toole T. E., Ylänne J., Shattil S. J., Ginsberg M. H. The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity. J Biol Chem. 1995 May 26;270(21):12411–12417. doi: 10.1074/jbc.270.21.12411. [DOI] [PubMed] [Google Scholar]
  19. Juliano R. L. Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu Rev Pharmacol Toxicol. 2002;42:283–323. doi: 10.1146/annurev.pharmtox.42.090401.151133. [DOI] [PubMed] [Google Scholar]
  20. Kimple R. J., De Vries L., Tronchère H., Behe C. I., Morris R. A., Gist Farquhar M., Siderovski D. P. RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity. J Biol Chem. 2001 May 31;276(31):29275–29281. doi: 10.1074/jbc.M103208200. [DOI] [PubMed] [Google Scholar]
  21. Knezevic I., Leisner T. M., Lam S. C. Direct binding of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both alphaIIb and beta3. J Biol Chem. 1996 Jul 5;271(27):16416–16421. doi: 10.1074/jbc.271.27.16416. [DOI] [PubMed] [Google Scholar]
  22. Kolanus W., Nagel W., Schiller B., Zeitlmann L., Godar S., Stockinger H., Seed B. Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell. 1996 Jul 26;86(2):233–242. doi: 10.1016/s0092-8674(00)80095-1. [DOI] [PubMed] [Google Scholar]
  23. Leung-Hagesteijn C. Y., Milankov K., Michalak M., Wilkins J., Dedhar S. Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin alpha-subunit-binding protein. J Cell Sci. 1994 Mar;107(Pt 3):589–600. [PubMed] [Google Scholar]
  24. Li Renhao, Mitra Neal, Gratkowski Holly, Vilaire Gaston, Litvinov Rustem, Nagasami Chandrasekaran, Weisel John W., Lear James D., DeGrado William F., Bennett Joel S. Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations. Science. 2003 May 2;300(5620):795–798. doi: 10.1126/science.1079441. [DOI] [PubMed] [Google Scholar]
  25. Liu Shouchun, Kiosses William B., Rose David M., Slepak Marina, Salgia Ravi, Griffin James D., Turner Christopher E., Schwartz Martin A., Ginsberg Mark H. A fragment of paxillin binds the alpha 4 integrin cytoplasmic domain (tail) and selectively inhibits alpha 4-mediated cell migration. J Biol Chem. 2002 Mar 27;277(23):20887–20894. doi: 10.1074/jbc.M110928200. [DOI] [PubMed] [Google Scholar]
  26. Lukashev M. E., Sheppard D., Pytela R. Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta 1 integrin cytoplasmic domain. J Biol Chem. 1994 Jul 15;269(28):18311–18314. [PubMed] [Google Scholar]
  27. Miyamoto S., Teramoto H., Coso O. A., Gutkind J. S., Burbelo P. D., Akiyama S. K., Yamada K. M. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol. 1995 Nov;131(3):791–805. doi: 10.1083/jcb.131.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Naik U. P., Patel P. M., Parise L. V. Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain. J Biol Chem. 1997 Feb 21;272(8):4651–4654. doi: 10.1074/jbc.272.8.4651. [DOI] [PubMed] [Google Scholar]
  29. O'Toole T. E., Katagiri Y., Faull R. J., Peter K., Tamura R., Quaranta V., Loftus J. C., Shattil S. J., Ginsberg M. H. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 1994 Mar;124(6):1047–1059. doi: 10.1083/jcb.124.6.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. O'Toole T. E., Mandelman D., Forsyth J., Shattil S. J., Plow E. F., Ginsberg M. H. Modulation of the affinity of integrin alpha IIb beta 3 (GPIIb-IIIa) by the cytoplasmic domain of alpha IIb. Science. 1991 Nov 8;254(5033):845–847. doi: 10.1126/science.1948065. [DOI] [PubMed] [Google Scholar]
  31. Papahadjopoulos D., Moscarello M., Eylar E. H., Isac T. Effects of proteins on thermotropic phase transitions of phospholipid membranes. Biochim Biophys Acta. 1975 Sep 2;401(3):317–335. doi: 10.1016/0005-2736(75)90233-3. [DOI] [PubMed] [Google Scholar]
  32. Patil S., Jedsadayanmata A., Wencel-Drake J. D., Wang W., Knezevic I., Lam S. C. Identification of a talin-binding site in the integrin beta(3) subunit distinct from the NPLY regulatory motif of post-ligand binding functions. The talin n-terminal head domain interacts with the membrane-proximal region of the beta(3) cytoplasmic tail. J Biol Chem. 1999 Oct 1;274(40):28575–28583. doi: 10.1074/jbc.274.40.28575. [DOI] [PubMed] [Google Scholar]
  33. Privalov P. L., Gill S. J. Stability of protein structure and hydrophobic interaction. Adv Protein Chem. 1988;39:191–234. doi: 10.1016/s0065-3233(08)60377-0. [DOI] [PubMed] [Google Scholar]
  34. Rojiani M. V., Finlay B. B., Gray V., Dedhar S. In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits. Biochemistry. 1991 Oct 15;30(41):9859–9866. doi: 10.1021/bi00105a008. [DOI] [PubMed] [Google Scholar]
  35. Saito Keigo, Sarai Akinori, Oda Masayuki, Azuma Takachika, Kozono Haruo. Thermodynamic analysis of the increased stability of major histocompatibility complex class II molecule I-Ek complexed with an antigenic peptide at an acidic pH. J Biol Chem. 2003 Feb 9;278(17):14732–14738. doi: 10.1074/jbc.M301086200. [DOI] [PubMed] [Google Scholar]
  36. Shattil S. J., O'Toole T., Eigenthaler M., Thon V., Williams M., Babior B. M., Ginsberg M. H. Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit. J Cell Biol. 1995 Nov;131(3):807–816. doi: 10.1083/jcb.131.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Simon K. O., Nutt E. M., Abraham D. G., Rodan G. A., Duong L. T. The alphavbeta3 integrin regulates alpha5beta1-mediated cell migration toward fibronectin. J Biol Chem. 1997 Nov 14;272(46):29380–29389. doi: 10.1074/jbc.272.46.29380. [DOI] [PubMed] [Google Scholar]
  38. Stevens J. M., Armstrong R. N., Dirr H. W. Electrostatic interactions affecting the active site of class sigma glutathione S-transferase. Biochem J. 2000 Apr 1;347(Pt 1):193–197. [PMC free article] [PubMed] [Google Scholar]
  39. Tudyka T., Skerra A. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein Sci. 1997 Oct;6(10):2180–2187. doi: 10.1002/pro.5560061012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ulmer Tobias S., Calderwood David A., Ginsberg Mark H., Campbell Iain D. Domain-specific interactions of talin with the membrane-proximal region of the integrin beta3 subunit. Biochemistry. 2003 Jul 15;42(27):8307–8312. doi: 10.1021/bi034384s. [DOI] [PubMed] [Google Scholar]
  41. Vallar L., Melchior C., Plançon S., Drobecq H., Lippens G., Regnault V., Kieffer N. Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein. J Biol Chem. 1999 Jun 11;274(24):17257–17266. doi: 10.1074/jbc.274.24.17257. [DOI] [PubMed] [Google Scholar]
  42. Vossmeyer D., Kaufmann C., Löster K., Lucka L., Horstkorte R., Reutter W., Danker K. The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif. Exp Cell Res. 2000 Apr 10;256(1):321–327. doi: 10.1006/excr.2000.4831. [DOI] [PubMed] [Google Scholar]
  43. Young B. A., Taooka Y., Liu S., Askins K. J., Yokosaki Y., Thomas S. M., Sheppard D. The cytoplasmic domain of the integrin alpha9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner. Mol Biol Cell. 2001 Oct;12(10):3214–3225. doi: 10.1091/mbc.12.10.3214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang X. A., Hemler M. E. Interaction of the integrin beta1 cytoplasmic domain with ICAP-1 protein. J Biol Chem. 1999 Jan 1;274(1):11–19. doi: 10.1074/jbc.274.1.11. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES