G-protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H₁-histamine or M₂-muscarinic receptors

Abstract

This paper describes a novel strategy to create a microarray of G-protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H₁-histamine receptor and the M₂-muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol-modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM-D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the ZeptoREADER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self-sorting liposome array of GPCRs which would underpin a variety of future novel applications.