Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor.
The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.
Jones
Brian W
BW
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Hinkle
Patricia M
PM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D016466:CHO Cells; D019556:COS Cells; D015220:Calcium Channels / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry / Q000502:physiology*; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; C000605255:Arrb1 protein, mouse; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D015220:Calcium Channels; D018076:DNA, Complementary; C506926:ITPR1 protein, human; D007074:Immunoglobulin G; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D010710:Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; D018025:Receptors, Thyrotropin-Releasing Hormone; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D013395:Sucrose; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11