systems are rapid and relatively easy to interpret, cellular assays have several
advantages:- they do not require any pre-purification of the receptor protein which make them
attractive for the screening of so-called orphan receptors whose cellular function and
physiological ligand have yet to be determined; - the conformation and hence the activity of the receptor protein is most likely to better
reflect the physiological situation than is the case for biochemical cell-free assays; - their use allows screening for potential therapeutic agents so as to quickly identify and
reject compounds that possess any cytotoxic properties; - they are readily adaptable to robotically controlledhigh-throughput screeningor
high-content screeningprotocols both of which are commonly based on fluorescence
microscopy using cells in microtitre plates (Section 18.2.3).
Much of the work using these assays is aimed at the discovery of new drugs and so
cells of human origin are the preferred targets. In practice such cells are expensive to
culture and are not readily adapted to automated assays. For such reasons, micro-
organisms are commonly used, with yeast being most commonly chosen because of
the high degree of conservation of basic molecular and cellular mechanisms between
yeast and human cells. Moreover, it is relatively easy to engineer yeast cells to
incorporate human receptor proteins such as GPCRs, RTKs and ion-channels.
Numerous analytical techniques have been used to probe signalling pathways
(Fig. 17.6). The majority are adaptations of standard techniques for studying
protein structure, protein–ligand interaction and protein–protein interaction. As a
result of their high sensitivity, fluorimetric techniques, particularly fluorescence
resonance energy transfer and fluorescence correlation spectroscopy, feature promin-
ently in these techniques. Techniques are now available for the site-specific post-
translational labelling of proteins with small fluorescent tags. Commercial companies
offer reagents, kits and services to facilitate the rapid identification of proteins
associated with the activation of a particular receptor. The most common approaches
include:- The use of monoclonal antibodies for western blots, protein purification and
immunocytochemistry. Western blots can identify changes in the expression of a
specific protein in the pathway; immunocytochemistry can detect movement within
the cell as a result of the activation of a pathway and the use of phosphospecific
antibodies can detect the phosphorylation of a particular protein. - The use ofknock-outandknock-instrategies using mice or cell lines, commonly
embryonic stem cells. In these techniques either the endogenous locus of the receptor
gene is manipulated or a modified receptor gene with an appropriate promoter is
expressed in the host. The effect of such action is then studied by techniques such as
Southern and western blots. - The use of microarray techniques – ‘chips’ are now commercially available on which
all the proteins of theSaccharomyces cerevisiaeproteome or a large proportion of
those of the human proteome have been individually deposited (see Sections 8.5.4
and 8.5.5 for further details). The approach is particularly suitable for identifying
686 Cell membrane receptors and cell signalling