title: The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes.
abstract: G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [(3)H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61±0.06 nM and 1.85±0.05 pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.67±0.05 nM and Bmax of 0.70±0.10 pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5-17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs.
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan; Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Arimitsu
authors.affiliation: Division of Cell-free Sciences, Proteo-Science Center, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Tomio
authors.initials: T
authors.lastname: Ogasawara
authors.affiliation: Division of Cell-free Sciences, Proteo-Science Center, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Division of Cell-free Sciences, Proteo-Science Center, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.affiliation: Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Hiasa
authors.affiliation: Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan. Electronic address: kazu@m.ehime-u.ac.jp.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Maeyama
mesh_terms: D000818:Animals; D001552:Benzazepines / Q000378:metabolism; D002474:Cell-Free System; D003342:Corpus Striatum / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008081:Liposomes; D008297:Male; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014908:Triticum
chemical_list: D001552:Benzazepines; D018492:Dopamine Antagonists; D008024:Ligands; D008081:Liposomes; D017447:Receptors, Dopamine D1; D011994:Recombinant Proteins; C534628:SCH 23390
keywords: Binding assay; Cell-free expression; Dopamine receptor; G-protein coupled receptors