Real-Time Analysis of G Protein-Coupled Receptor Signaling in Live Cells
Part of the
Methods in Molecular Biology™
book series (MIMB, volume 332)
Seven transmembrane-spanning receptors, widely referred to as G protein-coupled receptors (GPCRs), mediate a broad spectrum of extracellular signals at the plasma membrane through G proteins, thereby modulating a variety of biological processes. In addition to G proteins, they also interact with a number of other cytoplasmic proteins. Thus, methods to understand GPCR signaling and their interactions with intracellular proteins in real time in live cells are of importance. Recent developments in microscopy methods and the availability of fluorescent proteins facilitated the development of techniques to unravel these interactions more precisely. This chapter describes the methodology for sequential capturing of images of membrane and cytoplasmic proteins fused to different fluorescence probes to understand GPCR interaction with cytosolic proteins and their colocalization.
Key WordsG protein-coupled receptors live cell video microscopy green fluorescence protein red fluorescence protein internalization co-localization
Gether U. (2000) Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev.
, 90–113.PubMedCrossRefGoogle Scholar
Gether U., Asmar F., Meinild A. K., and Rasmussen S. G. (2002) Structural basis for activation of G-protein-coupled receptors. Pharmacol. Toxicol.
, 304–312.PubMedCrossRefGoogle Scholar
Pierce K. L., Premont R. T., and Lefkowitz R. J. (2002) Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol.
, 639–650.PubMedCrossRefGoogle Scholar
Lefkowitz R. J. (1998) G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J. Biol. Chem.
, 18,677–18,680.PubMedCrossRefGoogle Scholar
Shenoy S. K. and Lefkowitz R. J. (2003) Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signaling. Biochem. J.
, 503–515.PubMedCrossRefGoogle Scholar
Goodman O. B., Jr., Krupnick J. G., Santini F., et al. (1996) Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature
, 447–450.PubMedCrossRefGoogle Scholar
Brady A. E. and Limbird L. E. (2002) G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal.
, 297–309.PubMedCrossRefGoogle Scholar
Premont R. T. and Hall R. A. (2002) Identification of novel G protein-coupled receptor-interacting proteins. Methods Enzymol.
, 611–621.PubMedCrossRefGoogle Scholar
Galliera E., Jala V. R., Trent J. O., et al. (2004) β-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6. J. Biol. Chem.
, 25,590–25,597.PubMedCrossRefGoogle Scholar
Jala V. R., Shao W. H., and Haribabu B. (2005) Phosphorylation-independent beta-Arrestin Translocation and Internalization of Leukotriene B4 Receptors. J. Biol. Chem.
, 4880–4887.PubMedCrossRefGoogle Scholar