For a long time, hormone and neurotransmitter receptors remained abstract concepts whose existence was proposed only to explain pharmacological effects on target tissues. Since the mid-seventies, it has become possible to investigate of the receptor molecules themselves by the means of radioligand binding. This technique also allows the direct evaluation of the binding properties of any compound for a given membrane-bound receptor. Very often, radioligand binding experiments are performed with more-or-less purified cell membranes (Figure 26).
Radioligand binding is initiated by the incubation of cells, cell homogenates or purified plasma membrane preparations with an adequate radioactively labelled drug; the 'radioligand'. Adequate radioligands can be selected out of the wide variety of commercially available agonists and antagonists. Obviously, these radioligands should display high affininty and selectivity towards the receptor of interest. If no such radioligands are available, ligands can be custom-labelled by the investigator (for radioiodination) or by specialized institutions:
• Tritium [3H] and iodine [125I] are the most frequently used isotopes. Because of the long half-life of tritium (12.3 years), the tritiated ligand does not have to be resynthesized or repurchased frequently. They can be stored for a rather long time but, nevertheless, care should be taken to check for radiation-induced ligand degradation. In addition, because of the relatively low specific radioactivity of tritium (29 Ci/mmol), tritiated ligands are only suitable when the biological material contains sufficient amounts of the desired receptor.
• If not, radioiodinated ligands are more suitable because of the relatively high specific radioactivity of 125I (2125 Ci/mmol). However, the short half-life (60 days), the exposure of the investigator to gamma rays and the fact that the pharmacological and physicochemical properties of the iodinated ligand may deviate considerably from those of the original ligand constitute major drawbacks of this isotope.
G Protein- Coupled Receptors: Molecular Pharmacology From Academic Concept to Pharmaceutical Research Georges Vauquelin and Bengt von Mentzer © 2007 John Wiley & Sons, Ltd. ISBN: 978-0-470-51647-8
Separation of free and receptor-bound drug represents the most delicate step. This is commonly done by one of the three following techniques (Figure 27):
• Filtration: the free radioligand passes through the filter whereas the receptor-bound radioligand remains on the filter. Counting the radioactivity on the
filter allows the amount of receptor-bound radioligand to be quantified. This technique is usually employed when using membrane preparations and when performing radioligand binding to intact cells in suspension. The popularity of this technique results from the ability to handle a large number of samples with relative ease as well as the commercial availability of a variety of filtration devices. Moreover, the filters can be washed thoroughly and rapidly with fresh buffer (preferentially ice-cold to prevent dissociation of the radioligand-receptor complex). This allows the removal of remaining traces of free radioligand. The filters are usually of glass fibre, but sometimes it is also necessary to coat them with polyethyleneimine or to siliconize them to prevent radioligand absorption to the filter. For 'high throughput screening', the radioligand binding may be performed in microtiter plates with 96, 384 or even more wells. After the incubation, the contents of the wells are filtered simultaneously with a cell harvester. For modern high-throughput screening, robots are used to handle screen compounds and buffers as well as to perform the filtration step.
• Centrifugation: membranes or cells precipitate, whereas the free radioligand remains in solution, and can be discarded. Quantitation of the amount of receptor-bound radioligand is done by counting the radioactivity of the pellet. Since no thorough washing is involved, this technique is especially useful when the radioligand-receptor complex dissociates rapidly. However, this technique results in high background radioactivity due to the trapping of radioligand in the pellet. Manual manipulations and the resulting risk of contamination constitute additional disadvantages of the technique.
• Suction binding to intact cells may be achieved by plating them on the bottom of each well in (e.g. 24 well) multiwell plates. After the incubation, the free radioligand is removed by suction, the cells may then be washed with fresh buffer (preferentially ice-cold to prevent dissociation of the radioligand-receptor complex), and the remaining receptor-bound radioligand in each well is counted. For this purpose, plated cells are often treated with a detergent solution to solubilize the membranes. The radioactivity moves into solution and can then be counted easily. Here again, many manual manipulations are required.
The scintillation proximity assay (SPA) technique demands even fewer manipulations since the separation between free and bound radioligand is avoided (Figure 28). For this technique, small scintillant-containing beads are already present in the incubation tube/well. When these beads are also coated with wheat germ agglutinin (WGA), they will attach intact cells or membranes. The principle of the technique is based on the assumption that the overwhelming majority of the free radioligand molecules are too far from the beads for the scintillant to be activated whereas the receptor-bound radioligand is in close proximity to the beads and, hence, capable of stimulating the scintillant. Therefore, the measured scintillation will mainly arise from bound radioligand molecules.
Binding of a radioligand to a physiologically relevant receptor (i.e. 'specific binding') should at least obey the following criteria:
• The binding should be saturable, since a finite number of receptors are expected in a biological preparation.
• The potency of unlabelled ligands to compete with the radioligand for binding to the receptor should parallel their potency to provoke (for agonists) or block (for antagonists) receptor-mediated responses.
Radioligands not only bind to their receptor (Figure 29). They might also bind to other receptors and to non-receptor sites such as carrier proteins, enzymes, cell components recognizing certain chemical moieties of the radioligand (e.g. the catechol moiety for radiolabelled catecholamines). In addition, they might even radioligand
Specific binding Non-specific binding radioligand
Specific binding Non-specific binding
total binding — non-specific binding = specific binding
Figure 30 Determination of total and non-specific binding and the calculation of specific binding.
bind to separation materials such as filters or test tubes. This binding is called 'nonspecific binding'. One of the major problems in developing a suitable binding assay is the selection of a radioligand that shows enough specificity towards the receptor. In general, a hydrophilic (to avoid partitioning in the lipid bilayer of the membrane) radioligand with high affinity for the desired receptor may be a good candidate. However, some of the measured binding will always be non-specific. To deal with this problem, radioligand binding experiments always comprise two determinations: total binding and non-specific binding and the non-specific binding must be subtracted from the total binding to obtain the specific binding; i.e. binding to the receptor of interest (Figure 30).
Obtaining a correct non-specific binding value constitutes the most delicate aspect of a radioligand binding technique. In theory, non-specific binding can simply be obtained by adding an excess of competitor to the incubation mixture, so that binding of the radioligand to the receptors is completely displaced. In practice, care must be taken to choose a competitor that displaces the radioligand from the receptor only, and not from the other, non-specific sites. It is recommended to choose a potent competitor whose chemical structure is quite distinct from that of the radioligand.
Radioligand binding studies provide three main categories of information: saturation binding data, competition binding data and kinetic data.
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