G protein-coupled receptors: Evolution of ligand-mediated gating as regulator of binding affinity

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G protein-coupled receptors (GPCRs) are one of the largest groups of molecules transducing signals into nerve cells, and as such are the major pharmaceutical target for many human diseases and disorders, including depression, drug addiction, and neurodegenerative conditions - all of which increase in relevance for society and become more prevalent. However, despite the central importance of GPCRs, it is still unclear how their binding partners (ligands) access the active site which is often buried inside the GPCR molecules. Importantly, we have recently observed for an olfactory GPCR that its ligand possesses - in addition to the canonical inner binding site - a second binding site in a vestibule on the external surface of the GPCR, which blocks the passage of the ligand towards the inner binding site, thereby gating the receptor (ligand-gating) and decreasing its effective binding strength. We have in preliminary experiments already shown the presence of such vestibules in two neurotransmitter receptors and one other olfactory receptor. From these results we derive our central hypothesis: gating of ligand access via an external binding site (vestibule) constitutes an ancient and conserved mechanism to fine- tune binding affinity of GPCRs. The ultimate goal of the proposed project is to stringently examine this hypothesis. To that end we will now determine the presence and examine the function of a vestibule in a series of olfactory and neurotransmitter receptors chosen from several species including humans and the vertebrate model system zebrafish. First, we will identify vestibules in the selected receptors by computer-aided theoretical prediction of their structure using several independent methods. Second, we will introduce mutations in the amino acid sequence of receptors with predicted vestibular binding sites to eliminate these binding sites. Third, we will examine how binding of ligands is influenced in mutant receptors by expressing wild-type and mutant receptors in cells and visualizing the activation of the receptors through the subsequent signal transduction cascade. We predict that a successful completion of the proposed project will yield profound new insights into the basic mechanism of GPCR activation and could also have strong implications for targeted drug design, which is of critical importance in diseases such as depression, drug addiction, and neurodegenerative memory loss (serotonergic, cholinergic receptors, and dopaminergic receptors).