Probing the Binding Sites and Transmembrane Prolines of GPCRs Using Unnatural Amino Acids

Abstract

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation in functional G protein-coupled receptors (GPCRs). We have evaluated key aromatic residues in the binding sites of the M2 muscarinic acetylcholine receptor and the D2 and D4 dopamine receptors. In addition, highly conserved transmembrane proline residues of the D2 dopamine receptor have been probed with proline analogues and α-hydroxy acids. 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. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation-π 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-π interaction was found at the aligning residue in the M2 receptor. Incorporation of α-hydroxy acids at transmembrane proline sites 4.59, 5.50, 6.50, and 7.50 yielded D2 receptors with EC50 values similar to wild-type, whereas natural amino acids other than proline proved detrimental to receptor function. We propose that lack of hydrogen bond donor ability, common to both proline and α-hydroxy acids, is key to the functional role of proline within GPCR transmembrane helices.