The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes

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 D-1 receptors using a wheat cell -free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [H-3]SCH23390 to the synthesized receptors generated from a cell -free protein synthesis system or rat striatal membranes. From Scatcharcl plot analysis, the dissociation constant (K-d) and the maximum density (B-max) of the synthesized receptors were 6.61 +/- 0.06 nM and 1.85 +/- 0.05 pmolimg protein, respectively. The same analysis for rat striatal membrane gave a K-d of 2.67 +/- 0.05 nM and B-max, of 0.70 +/- 0.10 prnolfmg protein. Using a competition binding assay, K-i values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, buL K-i values of agonists, A68930, SKF38393 and dopamine, were 5-17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D-1 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 D-1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs. (C) 2014 Elsevier EN. All rights reserved.