Radioligand Binding Detection of Receptors in Brain Membranes

Abstract

Transmitters, hormones, and most of the therapeutic drugs exert their biological functions by generating signals in target cells through interaction with a receptor molecule in the cellular membrane, cytosol, or even at the nucleus of the cell. Because receptors play such a crucial role in cell function, especially within the central nervous system (CNS), we need lab techniques to measure their expression and functionality. There are techniques to determine regulatory changes in receptor number (receptor density), subcellular distribution, anatomical distribution, the physiological function of the receptors, as well as the ligand affinity.

Here we describe a classic way of doing this by exposing cells or tissue membranes to radiolabeled molecules (radioligands) that bind selectively to receptors in the sample (receptor binding assay). The functional consequence of the receptor occupancy or level of G-protein activation could be measured by [ ³⁵ S]GTPγS binding assay. This assay measures the binding of the non-hydrolyzable analogue [³⁵S]GTPγS ([³⁵S]guanosine-5′-O-(3-thio) triphosphate) to Gα subunits. [³⁵S]GTPγS binding assays, also known as functional binding assays, are carried out in a similar way as radioligand (receptor) binding assays.

The above radiometric assays are useful to differentiate among agonist, antagonist, and inverse agonist activities, in addition to traditional pharmacological parameters of potency and efficacy, as well as receptor constitutive activity and agonist-specific G-protein-coupled signaling. Moreover, radioligand binding assays provide a powerful tool for screening drug candidates for many receptors in drug discovery research.