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Structural Determinants of Pharmacological Specificity Between D₁ and D₂ Dopamine Receptors

Departments of Behavioral Neuroscience (K.A.N.) and Physiology and Pharmacology (H.L.), Oregon Health and Science University, and Veterans Affairs Medical Center (K.A.N.), Portland, Oregon; Department of Chemistry, University of California, Davis, California (M.M.T.); Department of Chemistry, Boston College, Chestnut Hill, Massachusetts (M.M.T.); and Department of Psychiatry, University of California Medical Center, Sacramento, California (C.J.D., M.M.T.)

To test the hypothesis that pharmacological differentiation between D₁ and D₂ dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D₂ receptor to the corresponding aligned residues in the D₁ receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D₂ receptors and 11 mutant D₁ receptors for D₁- and D₂-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D₁ receptor mutant W99^3.28F showed dramatically increased affinity for several D₂-selective antagonists, particularly spiperone (225-fold), whereas the D₂ receptor mutant Y417^7.43W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D₁-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V^5.39 caused modest but consistent and reciprocal changes in affinity of the receptors for D₁ and D₂-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D₁/D₂ receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

Address correspondence to: Kim A. Neve, Veterans Affairs Medical Center (R&D-30), 3710 SW U.S. Veterans Hospital Rd, Portland, OR 97239-2999. E-mail: nevek{at}ohsu.edu