Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies.
To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.
Jones
Brian W
BW
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Song
Gyun Jee
GJ
Greuber
Emileigh K
EK
Hinkle
Patricia M
PM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D054769:G-Protein-Coupled Receptor Kinase 2; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000378:metabolism; D009154:Mutation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D013997:Time Factors; D051552:beta-Adrenergic Receptor Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D000911:Antibodies, Monoclonal; C000605257:Arrb2 protein, rat; D019390:Arrestins; D006728:Hormones; D007295:Inositol Phosphates; D018025:Receptors, Thyrotropin-Releasing Hormone; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; C496569:Grk2 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2