Probing the role of the cation-pi interaction in the binding sites of GPCRs using unnatural amino acids.
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.
Torrice
Michael M
MM
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Bower
Kiowa S
KS
Lester
Henry A
HA
Dougherty
Dennis A
DA
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002412:Cations; D018389:Codon, Nonsense; D005461:Fluorine; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D043585:Receptor, Muscarinic M2 / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D055672:Static Electricity; D014981:Xenopus
D000596:Amino Acids; D002412:Cations; D018389:Codon, Nonsense; D043585:Receptor, Muscarinic M2; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D005461:Fluorine