G-Protein-Coupled Receptor Chromatographic Stationary Phases. 2. Ligand-Induced Conformational Mobility in an Immobilized β2-Adrenergic Receptor

Abstract

Membranes from a HEK-293 cell line expressing the β₂-adrenergic receptor (β₂-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 × 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (K_d) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized β₂-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile phase increased the retention of the CGP-12177A as did the addition of the agonist fenoterol. These results indicate that the immobilized β₂-AR retained its ability to undergo ligand-induced conformational changes. The data from this study suggest that the immobilized β₂-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor.