Abstract

G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 μg (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.

Highlights

  • The large-scale synthesis of functional G-protein-coupled protein receptors (GPCRs), which are known to be targets of various drugs (Lagerström and Schiöth, 2008), is a challenging experimental process

  • We showed that a wheat germ cell-free protein synthesis system, combined with glycerosomes, can be used to produce two functional GPCRs, namely wild-type HRH1 (WT-HRH1) and D107A-HRH1 on a large scale

  • We showed that the WTHRH1 proteoglycerosome generated by our wheat germ cell-free synthesis method selectively bound the histamine H1 antagonists and agonist and that the specific binding of [3H]pyrilamine to the synthesized WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased, but that this specific binding to the D107A mutant receptor proteoglycerosomes was weak, in contrast to the WT-HRH1 binding

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Summary

Introduction

The large-scale synthesis of functional G-protein-coupled protein receptors (GPCRs), which are known to be targets of various drugs (Lagerström and Schiöth, 2008), is a challenging experimental process. Investigation of the three-dimensional (3D) structures of GPCR molecules is a major objective of research as understanding the 3D structures is an essential prerequisite for developing target-directed drugs and rationally designed screening strategies, which are key approaches used in the pharmaceutical industry (Shimamura et al, 2011; Yinfeng et al, 2016). Most structural and functional approaches to the study of GPCRs require large-scale production of the receptor protein. Synthesizing a sufficient amount of functional GPCRs is limited by two main factors. GPCRs are membrane-bound proteins that favor a hydrophobic environment over a hydrophilic one, and synthesized GPCRs can aggregate rapidly in an aqueous reaction mixture

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