Dopamine D5 receptor agonist high affinity and constitutive activity profile conferred by carboxyl-terminal tail sequence.

Abstract The mammalian dopamine D1-like receptor gene family is comprised of two members, termed D1/D1A and D5/D1B. In an attempt to define the role of the carboxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacological and constitutive activity profiles, we examined a series of D5 receptor chimeras in which only the CT tail was swapped with corresponding sequences encoding human/vertebrate D1-like receptors. D5/D1CT or D5/D1DCT tail substitution mutants displayed a rank order of potency and agonist affinities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, similar to wt D1 receptors, did not exhibit receptor constitutive activity or responsiveness to inverse agonists. D1/D5CT or D1/D1DCT tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1CT and D5/D1DCT receptors. The affinities for numerous antagonists remained essentially unchanged for all receptor chimeras relative to parental wt receptors. A series of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438–448 and particularly Gln439, as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conserved D5/D1B receptor residue Gln439-(Ala/Ile), converts the full-length D5 receptor to one displaying “super” D5 characteristics with expressed affinities for discriminating agonists ∼4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP accumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino acid in this region can further regulate D5 agonist high affinity binding interactions independent of receptor constitutive activity.