Identification of amino acid determinants of dopamine 2 receptor synthetic agonist function

The human dopamine 2 receptor (hD2R) modulates locomotor activity, hormone secretion, and neuropsychiatric function. Current knowledge of the hD2R structure is in large part derived from mutagenesis studies and molecular pharmacologic analysis together with homology modeling using bovine rhodopsin as a template. In this study, we utilized comparison of the Drosophila D2-like receptor (DD2R) with the hD2R as a novel approach for identifying candidate amino acids that are determinants of ligand potency and/or efficacy. We focused our studies on four dopaminergic ligands that are used in the treatment of Parkinson's disease: bromocriptine, pergolide, piribedil, and ropinirole. All four ligands are potent agonists at the wild-type hD2R, whereas only bromocriptine shows comparable function at the DD2R. We performed site-directed mutagenesis to replace hD2R amino acids (modeled to project into the ligand binding pocket) with corresponding fly residues, and vice versa. Substitution of three amino acids in the hD2R with the homologous DD2R residues (V91A, C118S, and L170I) led to a pronounced loss of pergolide potency and efficacy. A converse triple amino acid substitution of human residues into the fly receptor (DD2R-A133V/S160C/I211L) markedly enhanced pergolide efficacy and potency at the mutant DD2R. The same substitutions also converted piribedil and ropinirole, which lacked appreciable activity on the DD2R, to partial agonists. These findings show the important role of these three residues in drug-receptor interactions. Our study illustrates that comparison of a mammalian receptor with an invertebrate homolog complements previously described strategies for defining G protein-coupled receptor structure-function relationships.