Screening of ligand binding sites of a G-protein coupled receptors by using photocrosslinking

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Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs), which can generally activate different signaling events. Recently developed polypeptide ligands of the corticotropinreleasing factor (CRF) receptor behaved as agonists and, at the same time, antagonists for the activation of different G-proteins. This finding implies significant differences between active conformations of the receptor and probably different ligand binding sites when coupled to different Gproteins. The structure-based development of signaling-selective ligands can result in a new type of drug candidates, but is hampered by a lack of high-resolution structures of GPCRs. We will use methods for site-directed incorporation of a photo-activatable amino acid (Bpa) into proteins to prepare a set of CRF receptor mutants bearing Bpa at various positions relevant for ligand binding. This will allow comparing ligands showing different selectivity for activation pathways with respect to their distinct binding sites. In course of this, we will find out whether Bpa-labeling of the receptor can confirm former results obtained with Bpa-labeled ligands, and we will verify the speculative ligand binding model for the CRF receptor, which is considered as common model for class B GPCRs.