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

The mammalian dopamine D1-like receptor gene family is comprised of two mem bers, termed D1/D1A and D5/D1B. In an attempt to define the role of the car boxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacol ogical and constitutive activity profiles, we examined a series of D5 recep tor chimeras in which only the CT tail was swapped with corresponding seque nces encoding human/vertebrate D1-like receptors, D5/D1(CT) or D5/D1D(CT) t ail substitution mutants displayed a rank order of potency and agonist affi nities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, simil ar to wt D1 receptors, did not exhibit receptor constitutive activity or re sponsiveness to inverse agonists, D1/D5(CY) or D1/D1D(CT) tail receptor mut ants displayed agonist pharmacological and functional characteristics not s ignificantly different from parental D1 or mutant D5/D1(CT) and D5/D1D(CT) receptors, The affinities for numerous antagonists remained essentially unc hanged for all receptor chimeras relative to parental wt receptors. A serie s of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438-448 and particularly Gln(439), as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conser ved D5/D1B receptor residue GLn(439)-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affin ities for discriminating agonists similar to 4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP ac cumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharm acological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino ac id in this region can further regulate D5 agonist high affinity binding int eractions independent of receptor constitutive activity.