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 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.

Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter.

Modification of the transport velocity of both the native neuronal and cloned presynaptic dopamine transporter (DAT) has been reported following activation/inhibition of second messenger system pathways. In order to identify the mechanism by which the functional activity of human DAT (hDAT) is regulated, we assessed the [3H]dopamine uptake kinetics, [3H] CFT binding characteristics, and, via immunofluorescent confocal microscopy, the cellular localization profiles of the hDAT expressed in both Sf9 and COS-7 cells following modulation of protein kinase C (PKC)- and protein kinase A (PKA)-dependent pathways. As with both native neuronal and cloned DATs, acute exposure of hDAT expressing Sf9 cells to the PKC activator PMA (1 microM), but not alphaPDD, reduced the Vmax (approximately 1 pmol/min/10(5) cells) for [3H]DA uptake by approximately 40%, an effect which was blocked by the protein kinase inhibitor staurosporine. Pretreatment of cells with staurosporine (500 nM) alone, however, increased [3H]DA uptake velocity by approximately 30%, an effect mimicked by the potent PKA inhibitor Rp-cAMPS. Activation of PKA-dependent pathways with Sp-cAMPS did not significantly modify DA uptake. Neither the Km of [3H]DA uptake (approximately 200 nM) nor the affinity of various substrates and transport inhibitors was altered by either PMA or staurosporine treatment. Despite changes in functional dopamine uptake velocity by PKC/PKA-dependent mechanisms, the estimated density of hDAT as indexed by whole-cell [3H] CFT binding was unchanged. Immunofluorescent confocal microscopy demonstrated that the observed functional consequence of PKC activation on [3H]DA uptake is associated with the rapid sequestration/internalization of hDAT protein from the cell surface, while the increase in DA uptake following PKC/PKA inhibition is the result of the recruitment of internalized or intracellular transporters to the plasma membrane. Identical rapid translocation patterns were observed in similarly treated COS-7 cells transiently expressing hDAT. These data suggest that the differential regulation of DAT transport capacity by both PKC- and PKA-dependent pathways are not a result of modifications in DAT catalytic activity. Moreover, the rapid shuttling of DATs between the plasma membrane and intracellular compartments provides an efficient means by which native DAT function may be regulated by second messenger systems, possibly following activation of presynaptic dopaminergic receptors, and suggests a role for cytoskeletal components in the dynamic regulation of DAT function.

A primordial dopamine D1-like adenylyl cyclase-linked receptor from Drosophila melanogaster displaying poor affinity for benzazepines.

We report here the isolation from Drosophila melanogaster of a 2.0 kb cDNA clone encoding a 385 amino acid protein (dDA1) displaying, within putative transmembrane domains, highest amino acid sequence homology (49-53%) to members of the vertebrate dopamine D1-like receptor family. When expressed in either Sf9 or COS-7 cells, dDA1 did not bind the specific D1-like receptor antagonist [3H]SCH-23390 or numerous other dopaminergic, adrenergic or serotoninergic ligands with high affinity. However, like vertebrate dopamine D1-like receptors, dDA1 stimulated the accumulation of cAMP in response to DA (EC50 approximately 300 nM) and 6,7-ADTN (EC50 approximately 500 nM). The dopaminergic rank order of potency (DA > NE >> 5-HT) and the lack of stimulation by other possible neurotransmitters (octopamine, tyramine, tryptamine) or DA metabolites (e.g. N-acetyl dopamine) found in Drosophila suggests that this receptor functionally belongs to the dopamine D1-like subfamily. Benzazepines, which characteristically bind to vertebrate dopamine D1-like receptors with high affinity, were relatively poor in stimulating (SKF-38393, SKF-82526; EC50 > 10 microM) dDA1-mediated accumulation of cAMP. Of the numerous compounds tested, a few dopaminergic antagonists inhibited DA-stimulated production of cAMP in a concentration-dependent manner, albeit with considerably reduced affinity, and with the rank order of potency: (+)-butaclamol(Kb approximately 125nM) > SCH-23390(Kb approximately 230nM) > alpha-flupenthixol (Kb approximately 400 nM) > chlorpromazine > or = spiperone (Kb approximately 680 nM) > or = clozapine. In situ hybridization revealed that dDA1 receptor mRNA is expressed as a maternal transcript, and at later blastoderm stages is restricted to apical regions of the cortical peripheral cytoplasm. The generation of inter-species D1 receptor chimeras may help to identify those particular sequence-specific motifs or amino acid residues conferring high affinity benzaepine receptor interactions.