If Rt = 1000 (the total number of Epo receptors per cell), Kd = 10"10 M, and [RL] = 100 (the number of Epo-occupied receptors needed to induce the maximal response), then an Epo concentration of 1.1 X 10"11 M will elicit the maximal response. If RT is reduced to 200/cell, then a ninefold higher Epo concentration (10"10 M) is required to occupy 100 receptors and induce the maximal response. If RT is further reduced to 120/cell, an Epo concentration of 5 X 10"10 M, a 50-fold increase, is necessary to generate the same cellular response.

Regulation of the number of receptors for a given signaling molecule expressed by a cell and thus its sensitivity to that signal plays a key role in directing physiological and developmental events. Alternatively, endocytosis of receptors on the cell surface can sufficiently reduce the number present to terminate the usual cellular response at the prevailing signal concentration.

Binding Assays Are Used to Detect Receptors and Determine Their Kd Values

Cell-surface receptors are difficult to identify and purify, mainly because they are present in such minute amounts. The receptor for a particular signaling molecule commonly con stitutes only = 10"6 of the total protein in the cell, or = 10"4 of the plasma-membrane protein. Purification is also difficult because these integral membrane proteins first must be sol-ubilized with a nonionic detergent so they can be separated from other proteins (see Figure 5-40).

Usually, receptors are detected and measured by their ability to bind radioactive ligands to cells or to cell fragments. The results of such a binding assay are illustrated and explained in Figure 13-4. Both the number of ligand-binding sites per cell and the Kd value are easily determined from the specific binding curve (Figure 13-4, curve B), which is described by Equation 13-2. Since each receptor generally binds just one ligand molecule, the number of ligand-binding sites equals the number of active receptors per cell. Straight binding assays like the one in Figure 13-4 are feasible with receptors that have a strong affinity for their ligands, such as the erythropoietin receptor (Kd = 1 X 10"10 M) and the insulin receptor on liver cells (Kd = 1.4 X 10"8 M).

Total binding A

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Total binding A

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