Dualsteric GPCR targeting: A novel route to binding and signaling pathway selectivity

Johannes Antony, Kerstin Kellershohn, Marion Mohr-Andra, Anna Kebig, Stefanie Prilla, Mathias Muth, Eberhard Heller, Teresa Disingrini, Clelia Dallanoce, Simona Bertoni, Jasmin Schrobang, Christian Trankle, Evi Kostenis, Arthur Christopoulos, Hans-Dieter Holtje, Elisabetta Barocelli, Marco De Amici, Ulrike Holzgrabe, Klaus Mohr

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Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M2-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M2 selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM2 cells disclosed pathway-specific signaling. Selective receptor activation (M2>M1>M3) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.-Antony, J., Kellershohn, K., Mohr-Andra, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Trankle, C., Kostenis, E., Christopoulos, A., Holtje, H.-D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.
Original languageEnglish
Pages (from-to)442 - 450
Number of pages8
JournalThe FASEB Journal
Publication statusPublished - 2009

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