Rho GTPases pp 37-58 | Cite as

Biochemical Assays to Characterize Rho GTPases

  • Mamta Jaiswal
  • Badri N. Dubey
  • Katja T. Koessmeier
  • Lothar Gremer
  • Mohammad R. AhmadianEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 827)


Rho GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, the GDP/GTP exchange and the GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the exchange of bound GDP for the cellular abundant GTP, GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to their inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators to understand the effect of binding on the protein structure and, ultimately, to gain insights into their biological functions. This chapter describes (1) detailed protocols for the expression and purification of Rho GTPases, of ­effector-binding domains, and catalytic domains of GEFs and GAPs; (2) the preparation of nucleotide-free and fluorescent nucleotide-bound Rho GTPases; and (3) methods for monitoring the intrinsic and GEF-catalyzed nucleotide exchange, the intrinsic and GAP-stimulated GTP hydrolysis, and the effector interaction with active GTPase (three alternative approaches).

Key words

Fluorescence spectroscopy GAP GEF GTPase Guanine nucleotide Mant Protein–protein interactions Rho Tamra Effector 


  1. 1.
    Ridley, A.J. (2001) Rho family proteins: coordinating cell responses. Trends Cell Biol 11, 471–477.PubMedCrossRefGoogle Scholar
  2. 2.
    Etienne-Manneville, S., and Hall, A. (2002) Rho GTPases in cell biology. Nature 420, 629–635.PubMedCrossRefGoogle Scholar
  3. 3.
    Burridge, K., and Wennerberg, K. (2004) Rho and Rac take center stage. Cell 116, 167–179.PubMedCrossRefGoogle Scholar
  4. 4.
    Raftopoulou, M., and Hall, A. (2004) Cell migration: Rho GTPases lead the way. Develop Biol 265, 23–32.PubMedCrossRefGoogle Scholar
  5. 5.
    Vetter, I.R., and Wittinghofer, A. (2001) The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304.PubMedCrossRefGoogle Scholar
  6. 6.
    Bishop, A.L., and Hall, A. (2000) Rho GTPases and their effector proteins. Biochem J 348, 241–255.PubMedCrossRefGoogle Scholar
  7. 7.
    Herrmann, C. (2003) Ras-effector interactions: after one decade. Curr Opin Struct Biol 13, 122–129.PubMedCrossRefGoogle Scholar
  8. 8.
    Dvorsky, R., Blumenstein, L., Vetter, I.R., and Ahmadian, M.R. (2004) Structural insights into the interaction of ROCKI with the switch regions of RhoA. J Biol Chem 279, 7098–7104.PubMedCrossRefGoogle Scholar
  9. 9.
    Moon, S.Y., and Zheng, Y. (2003) Rho GTPase-activating proteins in cell regulation. Trends Cell Biol 13, 13–22.PubMedCrossRefGoogle Scholar
  10. 10.
    Rossman, K.L., Der, C.J., and Sondek J. (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Revs Mol Cell Biol 6, 167–180.CrossRefGoogle Scholar
  11. 11.
    Dvorsky, R., and Ahmadian, M.R. (2004) Always look on the bright site of Rho − structural implications for a conserved intermolecular interface. EMBO Rep 5, 1130–1136.PubMedCrossRefGoogle Scholar
  12. 12.
    Ahmadian, M.R., Wittinghofer, A., and Herrmann, C. (2002) Fluorescence methods in the study of small GTP-binding proteins. Methods Mol Biol 189, 45–63.PubMedGoogle Scholar
  13. 13.
    Hemsath, L., and Ahmadian, M.R. (2005) Fluorescence approaches for monitoring interactions of RhoGTPases with nucleotides, regulators and effectors. Methods 37, 173–182.PubMedCrossRefGoogle Scholar
  14. 14.
    Eberth, A., and Ahmadian, M.R. (2009) In vitro GEF and GAP assays. In: Bonifacino, J.S, Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds) Current Protocols in Cell Biology, Unit 14.9. Wiley, New York.Google Scholar
  15. 15.
    Eberth, A., Dvorsky, R., Becker, C.F., Beste, A., Goody, R.S., and Ahmadian, M.R. (2005) Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins. Biol Chem 386, 1105–1114.PubMedCrossRefGoogle Scholar
  16. 16.
    Eberth, A., Lundmark, R., Gremer, L., Dvorsky, R., Koessmeier, K.T., McMahon, H.T,. and Ahmadian, M.R. (2009) A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family. Biochem J 417, 371–377.PubMedCrossRefGoogle Scholar
  17. 17.
    Ahmadian, M.R., Zor, T., Vogt, D., Kabsch, W., Selinger, Z., Wittinghofer, A., and Scheffzek, K. (1999) Guanosine triphosphatase stimulation of oncogenic Ras mutants. Proc Nat Acad Sci USA 96, 7065–7070.PubMedCrossRefGoogle Scholar
  18. 18.
    Hemsath, L., Dvorsky, R., Fiegen, D., Carlier, M.F., and Ahmadian, M.R. (2005) An electrostatic steering mechanism of Cdc42 recognition by Wiskott-Aldrich syndrome proteins. Mol Cell 20, 313–324.PubMedCrossRefGoogle Scholar
  19. 19.
    Stevens, W.K., Vranken, W., Goudreau, N., Xiang, H., Xu, P., and Ni, F. (1999) Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface. Biochem 38, 5968–5975.CrossRefGoogle Scholar
  20. 20.
    Haeusler, L.C., Blumenstein, L., Stege, P., Dvorsky, R., and Ahmadian, M.R. (2003) Comparative functional analysis of the Rac GTPases. FEBS Lett 555, 556–560.PubMedCrossRefGoogle Scholar
  21. 21.
    Fiegen, D., Haeusler, L.C., Blumenstein, L., Herbrand, U., Dvorsky, R., Vetter, I.R. and Ahmadian, M.R. (2004) Alternative splicing of Rac1 creates a self-activating GTPase. J Biol Chem 279, 4743–4749.PubMedCrossRefGoogle Scholar
  22. 22.
    Blumenstein, L., and Ahmadian, M.R. (2004) Models of the cooperative mechanism for Rho-effector recognition: Implications for RhoA-mediated effector activation. J Biol Chem 279, 53419–53426.PubMedCrossRefGoogle Scholar
  23. 23.
    Gremer, L., Merbitz-Zahradnik, T., Dvorsky, R., Cirstea, I.C., Kratz, C.P., Zenker, M., Wittinghofer, A., and Ahmadian, M.R. (2010) Impacts of germline mutations in KRAS on the GTPase cycle and effector interaction. Hum Mutat 32, 33–43.PubMedCrossRefGoogle Scholar
  24. 24.
    Gremer, L., De Luca, A., Merbitz-Zahradnik, T., Dallapiccola, B., Morlot, S., Tartaglia, M., Kutsche, K., Ahmadian M.R., and Rosenberger, G. (2010) Duplication of Glu37 in the switch I region of HRAS impairs effector/GAP binding and underlies Costello syndrome by promoting enhanced growth factor-dependent MAPK and AKT activation Hum Mol Genet 19, 790–802.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Mamta Jaiswal
    • 1
  • Badri N. Dubey
    • 1
  • Katja T. Koessmeier
    • 1
  • Lothar Gremer
    • 1
  • Mohammad R. Ahmadian
    • 1
    Email author
  1. 1.Medical Faculty, Institute of Biochemistry and Molecular Biology IIHeinrich-Heine UniversityDüsseldorfGermany

Personalised recommendations