The role of phosphorylation in D-1 dopamine receptor desensitization - Evidence for a novel mechanism of arrestin association

Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 03/2004; 279(9):7999-8010. DOI: 10.1074/jbc.M308281200
Source: PubMed

ABSTRACT Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D(1) receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.
    Molecular pharmacology 10/2008; 75(1):19-26. DOI:10.1124/mol.108.050542
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D(1) dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D(1) signaling is unclear. We now show that ethanol treatment of D(1) receptor-expressing cells decreases D(1) receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D(1) receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment. Ethanol was also found to modulate specific PKC activities as demonstrated using in vitro kinase assays where ethanol treatment attenuated the activities of lipid-stimulated PKCgamma and PKCdelta in membrane fractions, but did not affect the activities of PKCalpha, PKCbeta(1), or PKCvarepsilon. Importantly, ethanol treatment potentiated D(1) receptor-mediated DARPP-32 phosphorylation in rat striatal slices, supporting the notion that ethanol enhances D(1) receptor signaling in vivo. These findings suggest that ethanol inhibits the activities of specific PKC isozymes, resulting in decreased D(1) receptor phosphorylation and enhanced dopaminergic signaling.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 03/2008; 33(12):2900-11. DOI:10.1038/npp.2008.16
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently, we demonstrated that D(1) agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D(1) receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D(1) receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D(1) receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D(1) receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D(1) receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D(1) receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D(1) receptor that are distal from the ligand binding domain.
    Neuropharmacology 03/2007; 52(2):562-75. DOI:10.1016/j.neuropharm.2006.08.028

Daniel B Williams