PROJECT C6

Deciphering fast intra- and intermolecular dynamics of G protein-coupled receptors by Fluorescence (Cross) Correlation Spectroscopy

Project Description
Principal Investigator(s)
Publications


G-protein-coupled receptors (GPCRs) represent the most versatile and pharmacologically most important class of proteins that regulate cellular functions and constitute prominent targets for drug therapy. Although recently inactive as well as active X-ray structures have been solved, we know very little about the structural dynamics of their activation and deactivation.

This project aims to analyze the structural dynamics of GPCR activation. We will investigate the hypothesis that there are several rapid (<ms) activation steps that together constitute the activation dynamics of GPCRs beyond a two-state ‘on’-‘off’ model, and that ligands can influence these dynamics in distinct ways. We intend to resolve these rapid steps with a combined FRET-FCS approach (Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy). Structural fluctuations thereby will result in anticorrelated donor and acceptor signals due to the strong donor–acceptor distance dependence of FRET. The results are expected to provide valuable information about inter- and intramolecular dynamics of GPCR activation, notably the existence of multiple states and the kinetics of their transitions. We have developed fluorescence resonance energy transfer (FRET) techniques for GPCRs that allow the monitoring of the conformational change in GPCRs, which corresponds to agonist-induced activation. Even if it has been shown by us and others that typical activation times range between 30 and 50 ms, recent molecular modelling studies suggest movements that are about 3 orders of magnitude faster, based on microsecond binding of (low affinity) agonists to GPCRs and accommodating changes that lead to structural changes approaching the active state of GPCRs; The technique that covers the previously modelled fast dynamics as well as the already observed slower time scales is FCS. Recent approaches of FRET-FCS and technical advances such as interleaved dual-color excitation allow to ‘time-stamp’ donor and acceptor fluorescence with low background, and thus were proven to be a perfect method for studying structural fluctuations in proteins. By this FRET-FCS approach we expect to be able to distinguish kinetic components in the activation process, i.e. the potential occurrence of two or multiple intermediate activation states. In this context the FRET-FCS study, which will be related to kinetic FRET experiments from project C4, is expected to provide new insights into the dynamics time scale in GPCRs signalling.

Title Year Authors Journal Links
Kinetics and mechanism of G protein-coupled receptor activation 2014 Lohse, M.J., Maiellaro, I., and Calebiro, D. Curr Opin Cell Biol More
Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling 2012 Lohse, M.J., Nuber, S., and Hoffmann C. Pharmacol Rev More
Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1 2012 Hlavackova, V., Zabel, U., Frankova, D., Bätz, J., Hoffmann, C., Prezeau, L., Pin, J. P., Blahos, J., and Lohse, M. J. Sci Signal More
Conformational cross-talk between alpha2A-adrenergic and mu-opioid receptors controls cell signaling 2008 Vilardaga, J. P., Nikolaev, V. O., Lorenz, K., Ferrandon, S., Zhuang, Z., and Lohse, M. J. Nat Chem Biol More
Fluorescence correlation spectroscopy in living cells 2007 Kim, S.A., Heinze, K. G., and Schwille, P. Nat Methods More
Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry 2005 Kim, S.A, Heinze, K. G., Bacia, K., Waxham M.N., and Schwille, P. Biophys J More
A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells 2005 Hoffmann, C., Gaietta, G., Bünemann, M., Adams, S. R., Oberdorff-Maass, S., Behr, B., Vilardaga, J. P., Tsien, R. Y., Ellisman, M. H., and Lohse, M. J. Nat Methods More
Molecular basis of inverse agonism in a G protein coupled receptor 2005 Vilardaga, J. P., Steinmeyer, R., Harms, G. S. and Lohse, M. J. Nat Chem Biol More
Measurement of the millisecond activation switch of G protein-coupled receptors in living cells 2003 Vilardaga, J. P., Bünemann, M., Krasel, C., Castro, M., and Lohse, M. J. Nat Biotechnol More
Simultaneous Two-Photon Excitation of Distinct Labels for Dual-Color Fluorescence Cross-Correlation Analysis 2000 Heinze, K.G., Koltermann, A., and Schwille, P. Proc Natl Acad Sci USA More