GPCR-radioligand binding assays

Abstract

Radioligand binding assays provide sensitive and quantitative information about guanine nucleotide protein G protein-coupled receptor (GPCR) expression and affinity for a wide variety of ligands, making them essential for drug structure-activity studies and basic GPCR research. Three basic radioligand binding protocols, saturation, indirect (competition, displacement, or modulation), and kinetic binding assays, are used to assess GPCR expression (Bmax), equilibrium dissociation constants for radioligands (Kd) and nonradioactive ligands (Ki), association and dissociation rates, and to distinguish competitive and allosteric mechanisms of GPCR-ligand interactions. Nonspecific radioligand binding may be mitigated by appropriate choices of reaction conditions. Radioligand depletion (bound radioactivity >10% of total radioligand), which compromises accuracy of Kd and Ki measurements, can be limited by adjusting receptor concentration and appropriate radioligand choice. Accurate Kd and Ki values in saturation and indirect binding assays depend on binding equilibrium. Equilibration time for high-affinity ligands, with slow dissociation rates, may require much extended incubation times or increased incubation temperature.

Keywords: Binding equilibrium; Indirect binding; Ligand depletion; Radioligand binding; Radioligand binding artifacts; Saturation binding.