Preparing, measuring and capturing G-protein coupled receptor (GPCR) signalling complexes for future development of cell-free assay technologies

Author: Olgatina Bucco

Bucco, Olgatina, 2006 Preparing, measuring and capturing G-protein coupled receptor (GPCR) signalling complexes for future development of cell-free assay technologies, Flinders University, School of Medicine

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G-protein coupled receptors (GPCRs) are integral membrane proteins which represent primary cellular targets for intracellular signalling. Many of these receptors are altered in disease states and hence are the target for over 50% of marketed drugs. Despite their physiological importance, high-throughput, cell-free assays which measure functional or signalling activity are only recently being investigated. The current approach by the pharmaceutical industry to initially screen compounds for functionality is to use heterologous cell-based assay formats. The aim of this work was to reconstitute a cell-free GPCR signalling system on an appropriate platform (surface) as a prototype for future rapid drug screening and other applications. The proof-of-concept approach involved using the á2A-adrenergic receptor (á2A-AR) containing cell membrane preparations as the model GPCR, reconstituted with a set of heterotrimeric G-proteins; Gái1 and â1ă2 (the signal transducing complex being termed a “transductosome”). However, other receptors and G-proteins were also investigated. Receptors were initially obtained from natural (tissue) sources, however in the later stages they were expressed in a heterologous system (insect or mammalian expression system). G-proteins were expressed in Spodoptera frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor expression was verified by radioligand binding assays and endogenous G-proteins were removed from membrane preparations using the chaotropic agent urea to allow for reconstitution with purified G-proteins. Signal transduction through the transductosome was measured using the [35S]GTPăS binding assay. Receptor activated [35S]GTPăS binding was used to determine functional reconstitution and to validate that the system was working in the normal physiological manner both on and off a surface (with surface attachment being via histidine attachment on the Gái1 (6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent á2-AR antagonists), Prazosin (potent á1- AR antagonist) and Propranolol (â-AR antagonist) displayed the appropriate rank order for this class of receptor. This cell-free, surface-attached signalling complex prototype may have use in the future development of drug screening and discovery assay technologies as well as other applications as an alternative to cell-based assays which are not readily amendable to miniaturisation, long term storage and therefore stable robust microarray formats.

Keywords: GPCR,G-protein,Assay platform

Subject: Medical Biochemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2006
School: School of Medicine
Supervisor: Associate Professor EJ McMurchie