Determining the Activation of Rho as an Index of Receptor Coupling to G12/13 Proteins

  • Michio Nakaya
  • Mina Ohba
  • Motohiro Nishida
  • Hitoshi KuroseEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 746)


Heterotrimeric G proteins are composed of α, β, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by their cognate ligand, interaction with specific subtypes of G protein leads to dissociation of the α subunit of the heterotrimeric G protein from the βγ dimer, and both Gα-GTP and Gβγ are capable of initiating their own signal transduction pathways. G proteins are functionally divided into four groups based on the nature of α subunit into Gs, Gi, Gq, and G12 families. The members of the G12 subfamily are G12 and G13. Increasing evidence indicates that G12/13 proteins play critical roles in various physiological functions. G12 and G13 regulate the small GTPase Rho through modulation of guanine nucleotide exchange factor (RhoGEF) activity to regulate various cellular responses, such as cytoskeletal changes and cell growth. Therefore, Rho activity can often represent a sensitive marker of G12/13 activity. Here, we describe the Rho activation assay to monitor the activity of G12/13 proteins.

Key words

G12 Rho Pull-down Cardiomyocyte 


  1. 1.
    Northup, J.K., Smigel, M.D. and Gilman, A.G. (1982) The guanine nucleotide-activating site of the regulatory component of adenylate cyclase. J. Biol. Chem. 257, 11416–1423.PubMedGoogle Scholar
  2. 2.
    Benard, V., Bohl, B. P. and Bokoch, G. M., (1999) Characterization of Rac1 and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274, 13198–13204.PubMedCrossRefGoogle Scholar
  3. 3.
    Ren, X. D., Kiosses, W. B. and Schwartz, M. A. (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578–585.PubMedCrossRefGoogle Scholar
  4. 4.
    Yamaguchi, Y., Katoh, H. and Negishi, M. (2003) N-terminal short sequences of α-subunits of the G12 family determine selective coupling to receptors. J. Biol. Chem. 278, 14936–14939.PubMedCrossRefGoogle Scholar
  5. 5.
    Nishida, M., Tanabe, S., Maruyama, Y., Mangmool, S., Urayama, K., Nagamatsu, Y., Takagahara, S., Turner, J. H., Kozasa, T., Kobayashi, H., Sato, Y., Kawanishi, T., Inoue, R., Nagao, T. and Kurose, H. (2005) Gα12/13- and reactive oxygen species-dependent activation of c-jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J. Biol. Chem. 280, 18434–18441.PubMedCrossRefGoogle Scholar
  6. 6.
    Kurose, H. (2003) Gα12 and Gα13 as key regulatory mediator in signal transduction. Life Sci. 74, 155161.PubMedCrossRefGoogle Scholar
  7. 7.
    Worzfeld, T., Wettschureck, N. and Offermanns, S. (2008) G12/G13-mediated signalling in mammalian physiology and disease. Trends Pharmacol. Sci. 29, 582–589.PubMedCrossRefGoogle Scholar
  8. 8.
    Suzuki, N., Hajick, N. and Kozasa, T. (2009) Regulation and physiological functions of G12/13-mediated signaling pathways. Neurosignals 17, 55–70.PubMedCrossRefGoogle Scholar
  9. 9.
    Nishida, M., Sato, Y., Uemura, A., Narita, Y., Tozaki-Saitoh, H., Nakaya, M., Ide, T., Suzuki, K., Inoue, K., Nagao, T. and Kurose, H. (2008) P2Y6 receptor-Gα12/13 signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis. EMBO J. 27, 3104–3115.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Michio Nakaya
  • Mina Ohba
  • Motohiro Nishida
  • Hitoshi Kurose
    • 1
    Email author
  1. 1.Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical SciencesKyushu UniversityFukuokaJapan

Personalised recommendations