Nonconserved residues in the second transmembrane-spanning domain of the D₄ dopamine receptor are molecular determinants of D₄-selective pharmacology

The molecular determinants that govern selective ligand binding to the rat D₄ dopamine receptor were investigated by substituting D₂ dopamine receptor sequences into a D₄ dopamine receptor background. The resulting mutant D₄ dopamine receptors were then screened with a panel of 10 selective and nonselective ligands, which included two allosteric modulators as sensitive measures of protein conformational changes. Mutation of a phenylalanine at position 88 in the second transmembrane-spanning domain (TMS2) of the D₄ receptor to the corresponding valine in the D₂ receptor D₄-F88V resulted in an ~100-fold decrease in the affinity of the highly D₄-selective drug 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H- pyrrolo[2,3-b]pyridine (L-750,667) without substantially affecting the binding of the other ligands. Mutations at the extracellular side of D₄-TMS3 produced moderate decreases in L-750,667 binding affinities with concomitant increases in binding affinity for the D₂/D₃-selective antagonist (-)- raclopride. However, the binding affinities of these same D₄-TMS3 mutants for the allosteric modulator isomethylbutylamiloride also were an anomalous 6- to 20-fold higher than either wild-type receptor. In the combined D₄- F88V/FMS3 mutants, L-750,667 binding affinity was further decreased, but this decrease was not additive. More importantly, the combined D₄-F88V/FMS3 mutants had (-)-raclopride and isomethylbutylamiloride binding properties that reverted back to those of the wild-type D₄-receptor. In contrast to the D₄-F88V mutant, the adjacent D₄-L87W mutant had an increased affinity for ligands with extended structures but had essentially no effect on ligands with compact structures. These findings demonstrate that two residues near the extracellular side of D₄-TMS2 are critical molecular determinants for the selective binding of L-750,667 and ligands with extended structures.