Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes

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.

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…The reconstitution of proteins within the liposome bilayer typically proceeds by partially solubilising pre-formed ULVs with detergent, adding detergent solubilised protein and removing the detergent with bio-beads or dialysis to reform an intact bilayer containing the embedded protein. This has facilitated the study of a huge array of membrane proteins, including GPCRs in a controlled membrane environment [ 255 , 256 , 257 , 258 ].…”

Studying Membrane Protein–Lipid Specificity through Direct Native Mass Spectrometric Analysis from Tunable Proteoliposomes

“…This not only enables the detection of the membrane-embedded organization state but also enables us to titrate in/out specific membrane constituents/properties to tease apart their role in regulating the observed oligomeric organization states. Such customizability of liposomes to mimic and modulate properties of the native membrane has already made it the vehicle of choice to study a range of functions of membrane proteins, including ion transport, , G-protein driven signaling, , and protein trafficking, , to name a few. Very recently, there has also been a steady increase in the number of CryoEM structures of membrane proteins directly from liposomes. − Coupling these techniques with our liposome-nMS protocol will allow direct structural, functional, and mass spectral analysis of target membrane proteins directly from the same liposome samples.…”

Nucleic Acid Aptamers Emerging as Modulators of G-Protein-Coupled Receptors: Challenge to Difficult Cell Surface Proteins

“…Briefly, the authors developed aptamers against MRGPRX2, which is a crucial GPCR in non-IgE-dependent histamine release [ 30 ], by using liposomes to stabilize the GPCR produced from the wheat germ cell-free expression system. In the formation of the lipid bilayer, the authors used azolectin glycerosomes that contain a high concentration of glycerol in lipid particles to ensure the native fold of the GPCR [ 31 ]. For the selection of DNA aptamers, negative (counter) selection was conducted with mock liposomes lacking GPCR, and then positive selection was carried out with MRGPRX2-liposomes.…”