How does receptor dynamic influence ligand residence time and ligand efficacy at adenosine receptors

Project Description
Principal Investigator(s)

Ligand receptor interaction is often simply seen as a binding event and neglects the conformational dynamic of the target protein and its influence on ligand binding or the efficacy of the ligand. The central question within this project will be to address conformational dynamic of a receptor and monitor ligand binding with high kinetic resolution in living cells at the same time. We will use a fluorescence resonance energy transfer (FRET) based approach to address this question. Previously we have developed a generalized procedure which allows us to label G protein-coupled receptors at the C-terminus and third intracellular loop with two fluorophores. This approach allows us to monitor conformational changes of a receptor which occur upon ligand binding by intramolecular FRET. In particular it allows us to measure ligand on/off-rates and receptor activation/deactivation in real-time and will permit to obtain ligand residence time information and ligand efficacy data. We will combine such measurements with mutational analysis of the receptor protein to provide molecular insight into mechanisms of dynamic ligand binding, ligand efficacy, and the underlying influence on ligand residence time and vice versa.


Title Year Authors Journal Links
Optical probes based on G protein-coupled receptors - added work or added value? 2016 Stumpf, A.D.; Hoffmann, C. Br J Pharm More
beta-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle 2016 Nuber, S., Zabel, U., Lorenz, K., Nuber, A., Milligan, G., tobin, A. B., Lohse, M. J., and Hoffmann, C. Nature More
Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor 2014 Bock, A., Chirinda, B., Krebs, F., Messerer, R., Bätz, J., Muth, M., Dallanoce, C., Klingenthal, D., Tränkle, C., Hoffmann, C., De Amici, M., Holzgrabe, U., Kostenis, E., and Mohr, K. Nat Chem 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
The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling 2012 Bock, A., Merten, N., Schrage, R., Dallanoce, C., Bätz, J., Klöckner, J., Schmitz, J., Matera, C., Simon, K., Kebig, A., Peters, L., Muller, A., Schrobang-Ley, J., Tränkle, C., Hoffmann, C., De Amici, M., Holzgrabe, U., Kostenis, E., and Mohr, K. Nat Commun More
Comparison of the activation kinetics of the m3 acetylcholine receptor and a constitutively active mutant receptor in living cells 2012 Hoffmann, C., Nuber, S., Zabel, U., Ziegler, N., Winkler, C., Hein, P., Berlot, C. H., Bünemann, M., and Lohse, M. J. Mol Pharmacol More
Fluorescent labeling of tetracysteine-tagged proteins in intact cells 2010 Hoffmann, C., Gaietta, G., Zürn, A., Adams, S. R., Terrillon, S., Ellisman, M. H., Tsien, R. Y., and Lohse, Nat Protoc More
Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores 2010 Zürn, A., Klenk, C., Zabel, U., Reiner, S., Lohse, M. J., and Hoffmann, C. Bioconjug Chem More
Fluorescence Resonance Energy Transfer Analysis of {alpha}2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes 2009 Zürn, A., Zabel, U., Vilardaga, J. P., Schindelin, H., Lohse, M. J., and Hoffmann, C. Mol Pharmacol More
Molecular Basis of Partial Agonism at the Neurotransmitter {alpha}2A-Adrenergic Receptor and Gi-protein Heterotrimer 2006 Nikolaev, V. O., Hoffmann, C., Bünemann, M., Lohse, M. J., and Vilardaga, J. P. J Biol Chem 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