METHODS IN MOLECULAR BIOLOGY TM TM Volume 259 Receptor Signal Transduction Protocols SECOND EDITION Edited by Gary B Willars R A John Challiss Radioligand-Binding Methods for Membrane Preparations and Intact Cells David B Bylund, Jean D Deupree, and Myron L Toews Summary The radioligand-binding assay is a relatively simple but powerful tool for studying G-proteincoupled receptors There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined Detailed methods for typical radioligand-binding assays for G-protein-coupled receptors in membranes and intact cells are presented for these types of experiments Detailed procedures for analysis of the data obtained from these experiments are also given Key Words Affinity, assay, binding, competition, G-protein-coupled receptor, inhibition, intact cell, kinetic, nonspecific binding, radioligand, radioreceptor, rate constant, receptor, saturation Introduction The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors It can be used to determine the affinity of numerous drugs for these receptors, and to characterize regulatory changes in receptor number and in subcellular localization As a result, this assay is widely used (and often misused) by investigators in a variety of disciplines Our focus in this chapter is on radioligand-binding assays in membrane preparations from tissues and cell lines, and in intact cells Similar techniques, however, can be used to study solubilized receptors, receptors in tissue slices (receptor autoradiography), or receptors in intact animals From: Methods in Molecular Biology, vol 259, Receptor Signal Transduction Protocols, 2nd ed Edited by: G B Willars and R A J Challiss © Humana Press Inc., Totowa, NJ Bylund et al Fig Typical saturation experiment In this simulation the Bmax (receptor density) is 10 pM and the Kd (the dissociation constant or the free concentration that gives halfmaximal binding) is 100 pM There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity (Kd) of the radioligand for the receptor and the binding site density (Bmax) can be determined; (2) inhibition experiments from which the affinity (Ki) of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic experiments from which the forward (k+1) and reverse (k–1) rate constants for radioligand binding can be determined This chapter presents methods for typical radioligand-binding assays for G-protein-coupled receptors 1.1 Saturation Experiment Saturation experiments are frequently used to determine the change in receptor density (number of receptors) during development or following some experimental intervention, such as treatment with a drug A saturation curve is generated by holding the amount of receptor constant and varying the concentration of radioligand From this type of experiment the receptor density (Bmax) and the dissociation constant (Kd) of the receptor for the radioligand can be estimated The results of the saturation experiment can be plotted with bound (the amount of radioactive ligand that is bound to the receptor) on the y-axis and free (the free concentration of radioactive ligand) on the x-axis As shown in Fig 1, as the concentration of radioligand increases the amount bound Radioligand-Binding Assays Fig Typical inhibition experiment In this simulation the specific binding is 900 cpm and the IC50 (the concentration of drug that inhibits 50% of the specific binding) is 10 nM increases until a point is reached at which more radioactive ligand does not significantly increase the amount bound The resulting graph is a rectangular hyperbola and is called a saturation curve Bmax is the maximal binding which is approached asymptotically as radioligand concentration is increased Bmax is the density of the receptor in the tissue being studied Kd is the concentration of ligand that occupies 50% of the binding sites 1.2 Inhibition Experiment The great utility of inhibition experiments is that the affinity of any (soluble) compound for the receptor can be determined Thus these assays are heavily used both for determining the pharmacological characteristics of the receptor and for discovering new drugs using high-throughput screening techniques In an inhibition experiment, the amount of an inhibitor (nonradioactive) drug included in the incubation is the only variable, and the dissociation constant (Ki) of that drug for the receptor identified by the radioligand is determined A graph of the data from a typical inhibition experiment is shown in Fig The amount of radioligand bound is plotted vs the concentration of the unlabeled ligand (on a logarithmic scale) The bottom of the curve defines the amount of nonspecific binding The IC50 value is defined as the concentration of an unlabeled drug required to inhibit specific binding of the radioligand by 50% The Ki is then calculated from the IC50 Bylund et al Fig Typical association experiment In this simulation steady state is reached after approx 25 and lasts until the end of the experiment (42 min) 1.3 Kinetic Experiments Kinetic experiments have two main purposes The first is to establish an incubation time that is sufficient to ensure that steady state (commonly called equilibrium) has been reached The second is to determine the forward (k+1) and reverse (k–1) rate constants The ratio of these constants provides an independent estimate of the Kd (k–1/k+1) If the amounts of receptor and radioligand are held constant and the time varied, then kinetic data are obtained from which forward and reverse rate constants can be estimated A graph of the data from a typical association kinetic experiment is shown in Fig Initially the rate of the forward reaction exceeds the rate of the reverse reaction After approx 25 the amount of specific binding no longer increases and thus steady state has been reached From these data, the k+1 can be calculated For a dissociation experiment, the radioligand is first allowed to bind to the receptor and then the dissociation of the radioligand from the receptor is monitored by the decrease in specific binding (Fig 4) The rebinding of the radioligand to the receptor is prevented by the addition of a high concentration of a nonradioactive drug that binds to the receptor and thus blocks the receptor binding site, or by “infinite” dilution which reduces the free concentration of the radioligand Dissociation follows first-order kinetics and thus k–1 is equal to the t1⁄2 for dissociation divided by 0.693 (natural logarithm of 2) Radioligand-Binding Assays Fig Typical dissociation experiment In this simulation the t ⁄ (the time at which the specific binding has decreased by 50%) is 1.4 Assays in Intact Cells Although isolated membranes are by far the most common preparation used for radioligand-binding assays, for some purposes it is preferable to use intact cells The most obvious advantage of assays with intact cells is that the receptor is being studied in its native environment in the cell A related advantage of intact cell assays is that the binding properties of the receptor can be assessed in the same preparation and under essentially the same conditions as the functional responses mediated by the receptor are measured This allows a more direct comparison of the receptor binding properties with a wide variety of physiological responses following activation or inhibition of the receptor Intact cell assays may also be advantageous when a large number of different cell samples need to be studied, because intact cell assays eliminate the need to lyse cells and isolate membranes prior to assay For example, intact cell assays have proven very useful for preliminary screening of cell colonies following transfection with cDNA for various G-protein-coupled receptors, thus allowing rapid identification of clones for amplification and further analysis Most of the considerations that make intact cell assays advantageous in certain cases also represent limitations of intact cell assays in other cases For example, intact cell assays allow studies under physiological conditions, but they make it much more difficult to vary or control the assay conditions to Bylund et al identify factors that modulate receptor binding Radioligand uptake into cells by various transport processes can occur with intact cells, and care must be taken to ensure that radioligand association with intact cells is due to binding rather than uptake The occurrence of adaptive regulatory changes in receptor number, localization, and binding properties during the course of binding assays with intact cells can also present a serious complication (1) Finally, intact cells have membrane permeability barriers that are not present in isolated membrane preparations, and therefore the lipid solubility and membrane permeability of both the radioligand and the competing ligands must be considered in assays with intact cells Lipophilic (“lipid-loving”) ligands generally cross all cell membranes easily and thus have access to both cell surface receptors and those in intracellular compartments such as endosomes In contrast, hydrophilic (“water-loving”) ligands are relatively impermeable to the plasma membrane, and thus these ligands label only cell surface receptors Although these properties can complicate assays with intact cells, they also provide the basis for important radioligand-binding-based assays for receptor internalization, as discussed previously (1) Materials The information given in this section is specifically for assays with membrane preparations Additional information for intact cell assays is given in Subheading 3.4 A radioligand appropriate for the receptor being studied (see Note 1) For membrane saturation experiments, add the appropriate volume of radioligand into 550 µL of mM HCl in a glass test tube Thoroughly mix and add 200 µL of this solution to 300 µL of mM HCl Prepare successive dilutions in the same manner by adding 200 µL of each dilution to 300 µL of mM HCl to obtain the next lower dilution until six concentrations of radioligand have been prepared This dilution strategy gives a 100-fold range of radioligand concentrations Other dilution strategies will give different ranges as indicated in Table (see Note 2) For membrane inhibition and kinetic experiments, only a single concentration of radioligand is needed (see Note 3) A source of receptor, either membranes or intact cells The standard procedure for a membrane assay is to homogenize the tissue or cells of interest in a hypotonic buffer using either a Polytron (Brinkman) or similar homogenizer Remarkably, most receptors are stable at room temperature (generally for hours), although it is wise to put the tissue on ice quickly Homogenize about 500 mg of tissue in approx 35 mL of wash buffer (50 mM Tris-HCl or similar buffer at pH 7.0–8.0) using a Polytron (PT10-35 generator with PT10/TS probe) at setting for 20 s (see Note 4) The actual weight of tissue used should be recorded Centrifuge at 20,000 rpm (48,000g) in a Sorvall RC5-B using an SS34 rotor (or similar centrifuge and rotor) for 10 at 4° C (see Note 5) Decant the supernatant, and Radioligand-Binding Assays Table Dilution of Radioligand for Saturation Experiments µL of radioligand µL of diluent Dilution number 9 10 11 12 13 250.41 250.41 200.41 300.41 150.41 350.41 Relative concentration 100.78 150.78 125.78 112.58 116.28 113.18 111.68 110.78 110.39 100.41 14011 11611 116.41 112.61 111.01 110.41 10011 13011 119.01 112.71 110.81 110.24 repeat the homogenization and centrifugation The tissue preparation can either be used immediately or stored frozen as a pellet until needed (see Note 6) Generally protease inhibitors are not needed, but could be important in the case of certain tissues or with certain receptors Membrane assay buffer, 25 mM at pH 7.4, such as sodium phosphate or Tris For a few receptors the choice of buffer is important, but for most it is not Wash buffer such as 25 mM Tris, pH 7.4 Almost any buffer at neutral pH will serve the purpose mM HCl for diluting labeled and unlabeled ligands For many ligands, using a slightly acidic diluent will increase stability and decrease binding to test tubes Appropriate unlabeled ligands in solution 0.1 M NaOH for samples to be used to assay protein Polypropylene test tubes, 12 × 75 mm (assay tubes) Borosilicate glass test tubes, 12 × 75 mm (dilution tubes) Glass fiber filters (GF/A circles and GF/B strips) Filtration manifold Scintillation vials if using a 3H-radioligand, or test tubes if using a 125I-radioligand Scintillation cocktail (if using a 3H-radioligand) Methods 3.1 Saturation Experiment (Membrane Assay) Resuspend washed membrane preparation in distilled water by homogenization Add three 20-µL aliquots of the tissue suspension to 80 µL of 0.1 M NaOH for estimating protein concentration Bylund et al Add sufficient ice-cold assay buffer to the membrane suspension to give the appropriate final concentration (see Note 7) Set up a rack of 24 polypropylene incubation tubes, tubes across and tubes deep If using a 125I-ligand add two additional test tubes to each of the sets of tubes (for the determination of total added radioactivity) If using a 3H-ligand, prepare a set of 12 uncapped scintillation vials with GF/A glass fiber filter discs (see Note 8) To the 12 tubes on the last two rows, add 10 µL of a high concentration of an unlabeled ligand to determine nonspecific binding (see Note 9) To all 24 tubes add 970 µL of the membrane preparation Because this is a particulate suspension, it should be stirred slowly while aliquots are being removed Starting with the most dilute radioligand solution, add 20 µL to the columns of four tubes, and mix each tube Also add 20 µL of the radioligand solution to the two filter papers on the scintillation vials (if using a 3H-radioligand) or two test tubes (if using a 125I-ligand) for the determination of total added radioactivity Mix all the tubes again and incubate (usually at room temperature) for 45 Assuming that the system is at steady state, the exact time is not critical The tubes may need to be rearranged to be compatible with the specific style of filtration manifold used Filter the contents of the tubes and wash the filters twice with mL of wash buffer Depending on the rate of dissociation of the radioligand from the receptor, it may be important to use ice-cold wash buffer 10 Place the filters into scintillation vials, add mL of scintillation cocktail and cap if using a 3H-radioligand; or into test tubes if a using 125I-ligand 11 Shake the scintillation vials gently for h (or let stand at room temperature overnight) and then count in a liquid scintillation counter (if using a 3H-radioligand); or count in a gamma counter (if using a 125I-ligand) (see Note 10) 3.1.1 Calculation of Results from a Saturation Experiment Data from a sample saturation experiment are shown in Table (The methods and calculations for the sample competition and inhibition experiments are also available in an interactive format at http://www.unmc.edu/Pharmacology/ receptortutorial/.) Total binding and nonspecific binding can be plotted vs total added as shown in Fig for the sample experiment This plot allows one to detect data points that may be problematic Note that the nonspecific binding is linear (except possibly at the lowest concentrations), and that the specific binding saturates (is relatively constant) at high radioligand concentrations Specific binding is determined by subtracting nonspecific binding from total binding at each concentration of radioligand (see Table 2) The cpm values are converted to picomolar values using a conversion factor that accounts for specific activity for the radioligand, the counting efficiency of the particular scintillation counter used, and the conversion factor 2.2 × 1012 dpm/Ci For this experiment the counting efficiency was 0.36 and the specific radioactiv- Radioligand-Binding Assays Table Results of a Sample Saturation Experimenta in cpm Total added (cpm) 11,2360 11,5601 114,491 132,011 182,520 199,248 Total bound (cpm) Nonspecifically bound (cpm) Specifically boundb (cpm) 208 394 597 782 984 1210 18 25 46 88 189 416 190 369 551 694 795 794 [ H]RX821002 binding to human α2A-adrenergic receptors in HT-29 cells The amount of radioligand specifically bound was also determined by subtracting the nonspecifically bound from the total bound a b Fig Total binding and nonspecific binding vs total added for a sample experiment from Table ity of the radioligand was 60 Ci/mmol, and the factor for converting cpm to dpm is 0.0210 as shown: cpm dpm Ci mmol 1000 mL —— × ———– × —————— × —— × ———– × mL 0.36 cpm 2.2 × 1012 dpm 60 Ci L 2.10 × 10–2 pmol mol 1012 pmoL ————– × ————– = —————–—– 1000 mmol mol L The results of this conversion for the sample experiment are shown in Table 400 Freeman and Spina To make all possible pairs of comparisons following a significant ANOVA result (the usual situation, you have p < 0.05 in your initial ANOVA and now need a post hoc test to identify which groups are different), the Bonferroni test will give an answer, although it is not advisable to use this if you have more than five groups, as it is so conservative and thus has low power to detect differences Most texts recommend Tukey’s or Student–Newman–Keuls tests as being more powerful than Bonferroni for large numbers of comparisons, or the Ryan–Einot–Gabriel–Welsch procedure (10) Although widely used in the literature, Duncan’s multiple range test is not recommended for post hoc testing in statistics texts, because it has no means of controlling the pairwise error rate (10) If you have a very large number of complex comparisons or contrasts to make, then Sheffé’s method is probably the best to use, although rather conservative If you expect differences between groups to follow a specific order (e.g., in a dose–response or a time course), it is possible to apply a posttest for trend, rather like the procedure for linear regression, rather than testing pairs of groups This tests whether there is a trend for mean values to increase or decrease as you move through the groups 3.1.3 More Complex Designs of ANOVA One of the advantages of ANOVA as a statistical technique is its flexibility; it is possible to analyze complex experiments that make use of pairing, blockExample Posttest results for eosinophil data Because there are only four groups and the experimenter was interested in relatively few specific comparisons, that is, between Saline and Antigen, Saline and MAb A, Saline and MAb B, and MAb A vs MAb B, a Bonferroni test was used The relevant parts of the output (from SPSS) are shown below: Multiple comparisons Bonferroni Mean diff (I–J) I J Saline Antigen Antigen/MAb A Antigen/MAb B Ag/MAb A Antigen/MAb B Std error Sig 106.2* 93.4* 11.0 104.4* 6.533 6.533 6.533 6.533