A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

Manuel Grundmann, Irina G Tikhonova, Brian D Hudson, Nicola J Smith, Klaus Mohr, Trond Ulven, Graeme Milligan, Terry Kenakin, Evi Kostenis

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)


Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as the first sequential activator and corroborate its two-step activation in living cells by tracking integrated responses with innovative label-free biosensors that visualize multiple signaling inputs in real time. We validate this unique pharmacology with traditional cellular readouts, including mutational and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information.

Original languageEnglish
Pages (from-to)392-403
Number of pages12
JournalCell Chemical Biology
Issue number3
Publication statusPublished - 17 Mar 2016


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