title: 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/D1(CT) or D5/D1D(CT) 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/D5(CT) or D1/D1D(CT) tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1(CT) and D5/D1D(CT) 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 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 conserved D5/D1B receptor residue Gln(439)-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affinities for discriminating agonists approximately 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.
authors.affiliation: Departments of Psychiatry and Pharmacology and Institute of Medical Science, University of Toronto, Ontario, Canada M5S 1AB. l.demchyshyn@allelix.com
authors.forename: L L
authors.initials: LL
authors.lastname: Demchyshyn
authors.forename: F
authors.initials: F
authors.lastname: McConkey
authors.forename: H B
authors.initials: HB
authors.lastname: Niznik
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D018491:Dopamine Agonists / Q000378:metabolism*; D018492:Dopamine Antagonists / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050641:Receptors, Dopamine D5; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: C493605:DRD5 protein, human; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D008024:Ligands; D010446:Peptide Fragments; D017447:Receptors, Dopamine D1; D011993:Recombinant Fusion Proteins; D050641:Receptors, Dopamine D5; D000242:Cyclic AMP