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: 4.57). 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.

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    • "This data suggested that this C-terminal di-L motif is a plasma membrane targeting signal. Kim et al. [25] reported that the cell surface expression of a deletion mutant D1R (truncated at position 347) was diminished relative to the wild-type. In their mutant, di-L motif was intact, but the mutant could not get to the cell surface. "
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    ABSTRACT: The dopamine D₁ receptor (D₁R), a G protein-coupled receptor, plays a critical role in regulating blood pressure through its actions on renal hemodynamics and epithelial ion transport, which are highly linked to its intracellular trafficking. In this study, we generated a series of C-terminal mutants of D₁R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D₁R and cyclic AMP response to D₁R stimulation. D₁R with mutations within the endocytic recycling signal (amino acid residues 360-382) continued to be functional, albeit decreased relative to wild-type D₁R. Mutation of the palmitoylation site (347C>S) of D₁R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344-345L>A) by alanines resulted in the retention of D₁R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D₁R is critical for the glycosylation and cell surface targeting of D₁R.
    PLoS ONE 12/2011; 6(12):e29204. DOI:10.1371/journal.pone.0029204 · 3.23 Impact Factor
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    • "These and similar data obtained with a truncated opioid receptor [53] or with the leukotriene B4 receptor 1 (BLT1) [54] do not support a general role for receptor phosphorylation in the control of GPCRs internalization. Overall, these findings led Kim et al. to suggest that the role of receptor phosphorylation allow the access of arrestin to its receptor binding domain than to create an arrestin-binding site [55]. "
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    ABSTRACT: The process by which G protein-coupled receptors (GPCRs) are internalized through the clathrin-coated vesicles involves interactions of multifunctional adaptor proteins. These interactions are tightly controlled by phosphorylation and dephosphorylation mechanisms resulting in the regulation of receptor endocytosis. However, the identities of the kinases involved in this process remained largely unknown until recently. This paper discusses advances in our knowledge of the important role played by protein phosphorylation in the regulation of the endocytic machinery and how phosphorylation controls the coated vesicle cycle.
    International Journal of Cell Biology 06/2011; 2011(1687-8876):246954. DOI:10.1155/2011/246954
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    • "The binding of arrestin to GPCRs involves both phosphorylation-dependent and independent mechanisms. Phosphorylation of receptor intracellular domains by GPCR kinases may create a binding site for positively charged arrestin residues (Gurevich and Gurevich, 2006) and may also cause the intracellular domains to undergo conformational changes exposing nonphosphorylated residues that participate in forming the arrestin binding site (Kim et al., 2004). Receptor activation This work was supported by United States Public Health Service grants MH045372 (to K "
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    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 · 4.13 Impact Factor
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Daniel B Williams