Figure 2 (A) Binding of NPY to NPY receptor as a function of ligand concentration. Nonspecific binding was determined in the presence of X1000 excess of cold ligand. Specific binding was obtained by subtracting nonspecific binding from total binding. (B) Saturation binding of PYY to membranes prepared from CHO-K1 cells and CHO-K1 expressing NPY-Y2R. Inset shows the Scatchard plot from the saturation binding data.


Competition of binding to a specific ligand-receptor-binding site is determined using the radiolabeled ligand at about a concentration of Kd and a predetermined single concentration of various compounds from a compound library. The ''hits'' (compounds showing the required percentage inhibition) are confirmed by retesting. A full dose-response of the compounds at several concentrations (6-12 concentrations) is determined to obtain the IC 50 and the Kj for the hit compounds. Agonists and antagonists compete in the radioligand-binding to the receptor (they share the ability to bind to a common site on the receptor molecule) but differ in that antagonists are devoid of signal transduction activity. To differentiate a receptor-binding compound as agonist or antagonist, the compound has to be tested in an appropriate signal transduction assay. If the compound activates the signal in a functional assay, it is an agonist, and EC50 is determined. Neutral antagonists have no effect on basal receptor activity but inhibit the receptor signal transduction activity generated by a standard agonist for the receptor. Negative antagonists inhibit agonist-independent receptor activity and possess negative intrinsic activity.

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