Binding Assays Are Used to Detect Receptors and Determine Their Affinity and Specificity for Ligands
Receptors are usually detected and quantified by their ability to bind radioactively or fluorescently labeled ligands that have been added to the fluid surrounding intact cells or to cell fragments. This assay is based on the concept that the total number of receptors [RT] is equal to the number of free receptors [R] plus the number of ligand-bound receptors [RL].
The addition of increasing concentrations of ligand results in increasing amounts of cell-surface receptor–ligand complexes [RL]; as the concentration of ligand increases, the number of receptor-ligand complexes approaches, but never actually reaches, the total number of cell-surface receptors [RT]. In most cases, the amount of ligand added is vastly in excess of the amount of cell-surface receptors, so one can assume that the concentration of free ligand [L] is equal to the concentration of ligand added to the reaction. Thus, in these reactions, one need only measure the amount of ligand bound to surface receptors [RL] at each concentration of ligand added.
A plot of the amount of ligand bound to the receptor [RL] versus the amount of free ligand [L] generally follows equation 15-4, which is simply an algebraic transformation of equations 15-2 and 15-3. A typical ligand-binding curve can be seen in the red line in Figure 15-8.
FIGURE 15-8 Binding assays determine the Kd and the number of receptors per cell, but the maximal physiological response to an external signal usually occurs when only a fraction of the receptors are occupied by ligand. In a typical experiment to determine the affinity of a receptor for a ligand, radioactively or otherwise labeled ligand is incubated with cells that do not express the receptor of interest and with cells that have been altered by recombinant DNA techniques to express that receptor on their surface. Incubation is generally for an hour at 4 °C; the low temperature is used to prevent endocytosis of the cell-surface receptors. The cells are then separated from unbound ligand, usually by centrifugation and washing with buffer, and the amount of radioactivity bound to the cells is measured. “Background” binding by control cells is subtracted from the binding to the receptor-expressing cells, and the amount of bound ligand per cell is calculated and plotted (red curve) as a function of the ligand concentration. Note that even at relatively high ligand concentrations, the number of receptor-bound ligand molecules approaches, but does not equal, the number of cell-surface receptors. Nonetheless, by analysis of the data using equation 15-4, one can determine that these cells express 1000 receptors for this ligand, and that the Kd for binding of the ligand is 1 nM. In parallel experiments, the physiological response of the cell to increasing concentrations of ligand is also measured (blue line). Typically, the plots of the extent of ligand binding to the receptor and of physiological response at different ligand concentrations differ. In the example shown here, 50 percent of the maximal physiological response is induced at a ligand concentration at which only 18 percent of the receptors are occupied. Likewise, 80 percent of the maximal response is induced when the ligand concentration equals the Kd value, at which 50 percent of the receptors are occupied.
Computer curve-fitting programs are typically used to calculate the RT and Kd values. Using this approach, one can calculate from this binding curve that there are 1000 receptors per cell surface and that the Kd for binding this ligand is 1 × 10−9 M, or 1 nM.