 Abstract | 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 �2A-adrenergic receptor (�2A-AR)
containing cell membrane preparations as the model GPCR, reconstituted with a set
of heterotrimeric G-proteins; G�i1 and �1�2 (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]GTP�S binding assay. Receptor
activated [35S]GTP�S 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 G�i1
(6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent �2-AR antagonists), Prazosin (potent �1-
AR antagonist) and Propranolol (�-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. |
