17.3 Ligand-binding and cell-signalling studies
17.3.1 Selection of ligand and receptor preparation
Receptor preparations
Preparations of receptors for ligand-binding studies may either leave the membrane
intact or involve the disruption of the membrane and the release of the receptor with
or without membrane fragments, some of which could form vesicles with variable
receptor orientation and control mechanisms. Membrane receptor proteins show no or
very little ligand-binding properties in the absence of phospholipid so that if a
purified receptor protein is chosen, it must be introduced into a phospholipid vesicle
for binding study purposes. The range of receptor preparations available for binding
studies is shown in Table 17.2.
Kinetic studies aimed at the determination of individual rate constants are best
carried out using isolated cells whilst studies of the number of receptors in intact
tissue are best achieved by labelling the receptors with a radiolabel preferably using
an irreversible competitive antagonist and applying the technique of quantitative
autoradiography.Ligands
A common technique for the study of ligand–receptor interaction is the use of a
radiolabelled ligand with isotopes such as^3 H,^14 C,^32 P,^35 S and^135 I. Generally a high-
specific-activity ligand is used as this minimises the problem of non-specific binding
(see below). If a large number of ligands are being studied, such as in the screening of
potential new therapeutic agents, the cost and time of producing the radiolabelled
forms becomes virtually prohibitive and experimental techniques such as fluorescence
spectroscopy and surface plasmon resonance spectroscopy have to be used. However,
the use of radiolabelled ligands remains attractive as a means of distinguishing
between orthosteric and allosteric ligands.
The technique of using radiolabelled ligand generally requires the separation of
bound and unbound ligand once equilibrium has been achieved. This is most com-
monly achieved by techniques such as equilibrium dialysis and ultrafiltration exploit-
ing the inability of receptor-bound ligand to cross a semi-permeable membrane, and
by simple centrifugation exploiting the ability of the receptor-bound ligand to be
pelleted by an applied small centrifugal field.
For the study of ligand–receptor interactions that occur on a sub-millisecond
timescale, special approaches such as stopped-flow and quench-flow methods
(Section 15.3.3) need to be adopted to deliver the ligand to the receptor. An alternative
approach is the use of so-calledcaged compounds. These possess no inherent ligand
properties but onlaser flash photolysiswith light of a specific wavelength, a protect-
ing group masking a key functional group is instantaneously cleaved releasing the
active ligand.680 Cell membrane receptors and cell signalling