Biochemical Methods to Study the Interactions Between Integrins and Ion Channels

Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 674)


Protein-protein interactions between integrins and ion channels consist in a complicated bidirectional talk, not yet understood in detail, which triggers a downstream signaling network. Such a coordinated process occurs in discrete, localized microcompartments and involves different membrane and cytoplasmic proteins. Since the early nineties, when the first functional association between integrins and ion channels was characterized, the number of similar examples is constantly increasing. Identifying the components of this pathway has general importance for cell physiology and will eventually lead to fully understand the role of ion channels in the physiological processes typically controlled by integrin receptors, such as cell adhesion, migration and proliferation.

Here, we detail the main experimental methods currently available to study these processes and discuss their advantages and disadvantages. Biochemical copurification and genetic interaction studies, as well as high-throughput screening, can be performed to initially identify the interacting proteins. Successively, in vitro binding assays such as pull-down and immunoprecipitation-based techniques allow to verify and better characterize these partnerships, possibly in combination with mass spectrometry methods.

When transient interactions are involved, more sophisticated techniques, such as photoaffinity labeling procedures, are necessary to detect the multiprotein complexes by having them covalently bound together as they interact. To provide even more thorough analyses of the formation, function and composition of protein complexes, other technologies such as confocal microscopy, fluorescence resonance energy transfer microscopy and site directed mutagenesis (possibly in murine models) have to be performed.

The progressive accumulation of data defining novel protein-protein interactions has been considerably accelerated by the identification of specific sequence motifs that regulate integrin binding to other proteins as well as integrin recognition sequences in the ligand. Moreover, the availability of protein tagging strategies and the increased sensitivity of mass spectrometry-based methods for protein identification have also contributed important tools. In the near future, the coupling of traditional techniques with proteomic approaches is likely to offer invaluable help in unraveling integrin-ion channel interactions, thus elucidating the biological implication of these complexes.


Transient Receptor Potential Vanilloid Bait Protein Prey Protein Aryl Azide Label Transfer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Arcangeli A, Becchetti A. Complex functional interaction between integrin receptors and ion channels. Trends Cell Biol 2006; 16:631–9.CrossRefPubMedGoogle Scholar
  2. 2.
    Rouslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996; 12:697–715.CrossRefGoogle Scholar
  3. 3.
    Selbach M., Mann M. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 2006; 3:981–83.CrossRefPubMedGoogle Scholar
  4. 4.
    Morell M, Espargaró A, Avilés FX et al. Detection of transient protein-protein interactions by bimolecular fluorescence complementation: The Antibodyl-SH3 case. Proteomics 2007; 7:1023–36.CrossRefPubMedGoogle Scholar
  5. 5.
    Kotani N, Gu J, Isaji T et al. Biochemical visualization of cell surface molecular clustering in living cells. Proc Natl Acad Sci USA 2008; 105:7405–9.CrossRefPubMedGoogle Scholar
  6. 6.
    Phizicky E, Fields S. Protein-protein interactions: methods for detection and analysis. Microbiol Rev 1995; 59:94–123.PubMedGoogle Scholar
  7. 7.
    Van Criekinge W, Beyaert R. Yeast two-hybrid: state of the art. biol proced online (www.biologicalprocedures. com) 1999; 2:1–38.Google Scholar
  8. 8.
    Niethammer M, Sheng M. Identification of ion channel-associated proteins using the yeast two-hybrid system. Methods Enzymol 1998; 293:104–22.CrossRefPubMedGoogle Scholar
  9. 9.
    Guo D, Rajamäki ML, Valkonen. Protein-protein interactions: the yeast two hybrid system. Methods Mol Biol 2008; 451:421–39.CrossRefPubMedGoogle Scholar
  10. 10.
    Larkin D, Murphy D, Reilly DF et al. ICln, a novel integrin alpha(IIb)beta3-associated protein, functionally regulates platelet activation. J Biol Chem 2004; 279:27286–93.CrossRefPubMedGoogle Scholar
  11. 11.
    Lal A, Haynes SR, Gorospe M. Clean western blot signals from immunoprecipitated samples. Mol Cell Probe 2005; 19:385–8.CrossRefGoogle Scholar
  12. 12.
    Ransone LJ. Detection of protein protein interactions by coimmunoprecipitation and dimerization. Method Enzymol 1995; 254:491–7.CrossRefGoogle Scholar
  13. 13.
    Qoronfleh MW, Ren L, Emery D et al. Use of immunomatrix methods to improve protein-protein interaction detection, J Biomed Biotechnol 2003; 2003(5):291–8.CrossRefPubMedGoogle Scholar
  14. 14.
    McPhee JC, Dang YL, Davidson N et al. Evidence for a functional interaction between integrins and G protein-activated inward rectifier K+ channels. J Biol Chem 1998; 273; 34696–702.CrossRefPubMedGoogle Scholar
  15. 15.
    Ivanina T, Neusch C, Yong-Xin L et al. Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without β1 integrins. FEBS Letts 2000; 466:327–32.CrossRefGoogle Scholar
  16. 16.
    Levite M, Cahalon L, Peretz A et al. Extracellular K+ and opening of voltage-gated potassium channels activate T-cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins. J Exp Med 2000; 191:1167–76.CrossRefPubMedGoogle Scholar
  17. 17.
    Alessandri Haber N, Olayinka AD, Joseph EK et al. Interaction of transient receptor potential vanilloid 4, integrin and SRC tyrosine kinase in mechanical hyperalgesia. J Neurosci 2008; 28:1046–57.CrossRefPubMedGoogle Scholar
  18. 18.
    Cherubini A, Pillozzi S, Hofmann G et al. HERG K+ channels and β1 integrins interact through the assembly of a macromolecular complex. Ann NY Acad Sci 2002; 973:559–61.CrossRefPubMedGoogle Scholar
  19. 19.
    Cherubini A, Hofmann G, Pillozzi S et al. Herg1 channels are physically linked to beta 1 integrins and modulate adhesion-dependent signalling. Mol Biol Cell 2005; 16:2972–83.CrossRefPubMedGoogle Scholar
  20. 20.
    Abdel-Ghany M, Cheng HC, Elble RC et al. The interacting binding domains of the beta4 integrin and calcium-activated chloride channels (CLCAs) in metastasis. J Biol Chem 2003; 278:49406–16.CrossRefPubMedGoogle Scholar
  21. 21.
    Wyszynski M, Sheng M. Analysis of ion channels associated proteins. Methods Enzymol 1999; 294:371–385.CrossRefPubMedGoogle Scholar
  22. 22.
    Nooren IMA, Thornten MA. J Mol Biol 2003; 325:991–1018.CrossRefPubMedGoogle Scholar
  23. 23.
    Bomgarden RD. Studying protein interactions in living cells. Genetic Engineering and Biotechnology News ( 2008; 28(7).Google Scholar
  24. 24.
    Fancy D. Elucidation of protein-protein interactions using chemical cross-linking or label transfer techniques. Curr Opin Chem Biol 2000; 4:28–33.CrossRefPubMedGoogle Scholar
  25. 25.
    Häse CC, Minchin RF, Kloda A et al. Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli. Biochem Biophys Res Comm 1997; 232:777–82.CrossRefPubMedGoogle Scholar
  26. 26.
    Mahlknecht U, Ottmann OG, Hoelzer D. J Biotechnol 2001; 88:89–94.CrossRefPubMedGoogle Scholar
  27. 27.
    Abdel-Ghany M, Cheng HC, Elble RC et al. The breast cancer β4 integrin and endothelial human CLCA2 mediate lung metastasis. J Biol Chem 2001; 276:25438–46.CrossRefPubMedGoogle Scholar
  28. 28.
    Howell JM, Winstone TL, Coorssen JR et al. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins. Proteomics 2006; 6:2050–69.CrossRefPubMedGoogle Scholar
  29. 29.
    Arcangeli A, Becchetti A, Mannini A et al. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels. J Cell Biol 1993; 122:1131–43.CrossRefPubMedGoogle Scholar
  30. 30.
    Davis MJ, Wu X, Nurkiewicz TR et al. Regulation of ion channels by integrins. Cell Biochem Biophys 2002; 36:41–66.CrossRefPubMedGoogle Scholar
  31. 31.
    Hofmann G, Bernabei PA, Crociani O et al. HERG K+ channels activation during β1 Integrin-mediated Adhesion to fibronectin induces an up-regulation of αvβ3 Integrin in the preosteoclastic leukemia cell line FLG 29.1. J Biol Chem 2001; 276:4923–31.CrossRefPubMedGoogle Scholar
  32. 32.
    Waitkus-Edwards KR, Martinez-Lemus LA, Trzeciakovski JP et al. α4β1 integrin activation of L-type calcium channels in vascular smooth muscle causes arteriole vasoconstriction. Circ Res 2002; 90:473–80.CrossRefPubMedGoogle Scholar
  33. 33.
    Yang W, Steen H, Freeman MR. Proteomic approaches to the analysis of multiprotein signalling complexes. Proteomics 2008; 8:832–51.CrossRefPubMedGoogle Scholar
  34. 34.
    Bunnell SC, Hong DI, Kardon JR et al. T-cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J Cell Biol 2002; 58:1263–1275.CrossRefGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2010

Authors and Affiliations

  1. 1.Department of Experimental Pathology and OncologyUniversity of FirenzeItaly

Personalised recommendations