title: Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor.
abstract: Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [(125)I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449-453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM1 receptor and further determined the region of the CLR C-tail responsible for this GRK function.
authors.affiliation: Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692, Japan. Electronic address: kuwasako@med.miyazaki-u.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kuwasako
authors.affiliation: Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Sekiguchi
authors.affiliation: Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Nagata
authors.affiliation: Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692, Japan.
authors.forename: Danfeng
authors.initials: D
authors.lastname: Jiang
authors.affiliation: Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Murakami
authors.affiliation: Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Hattori
authors.affiliation: Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kitamura
authors.affiliation: Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692, Japan.
authors.forename: Johji
authors.initials: J
authors.lastname: Kato
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology; D054771:G-Protein-Coupled Receptor Kinase 4 / Q000378:metabolism*; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D058265:Receptors, Adrenomedullin / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D058265:Receptors, Adrenomedullin; D043562:Receptors, G-Protein-Coupled; D054771:G-Protein-Coupled Receptor Kinase 4; D054774:G-Protein-Coupled Receptor Kinase 5; C517855:GRK4 protein, human; C517898:GRK5 protein, human
keywords: Adrenomedullin; Calcitonin receptor-like receptor; Receptor activity-modifying protein 2; Receptor mutations; Receptor trafficking; Signal transduction