title: The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association.
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.
authors.affiliation: Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406, USA.
authors.forename: Ok-Jin
authors.initials: OJ
authors.lastname: Kim
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Gardner
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Williams
authors.forename: Paul S
authors.initials: PS
authors.lastname: Marinec
authors.forename: David M
authors.initials: DM
authors.lastname: Cabrera
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Peters
authors.forename: Chun C
authors.initials: CC
authors.lastname: Mak
authors.forename: Kyeong-Man
authors.initials: KM
authors.lastname: Kim
authors.forename: David R
authors.initials: DR
authors.lastname: Sibley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D001552:Benzazepines / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D017447:Receptors, Dopamine D1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014316:Tritium
chemical_list: D019393:Arrestin; D001552:Benzazepines; D008164:Luminescent Proteins; D017447:Receptors, Dopamine D1; D011994:Recombinant Proteins; D014316:Tritium; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP