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

John A. Schetz, Peter S. Benjamin, David R. Sibley

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29 Scopus citations


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.

Original languageEnglish
Pages (from-to)144-152
Number of pages9
JournalMolecular Pharmacology
Issue number1
StatePublished - 2000


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