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      Probing the role of the cation-pi interaction in the binding sites of GPCRs using unnatural amino acids.

      Proceedings of the National Academy of Sciences of the United States of America
      Amino Acids, chemistry, metabolism, Animals, Binding Sites, Cations, Codon, Nonsense, Fluorine, Humans, Ion Channel Gating, Models, Molecular, Protein Structure, Secondary, Receptor, Muscarinic M2, Receptors, Dopamine D2, Receptors, G-Protein-Coupled, Static Electricity, Xenopus

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          Abstract

          We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug-receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K(+) channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation-pi interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed "rotamer switch" models. Interestingly, no comparable cation-pi interaction was found at the aligning residue in the M2 receptor.

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