G-protein-coupled receptor chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor.
Membranes from a HEK-293 cell line expressing the beta(2)-adrenergic receptor (beta(2)-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 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 beta(2)-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 beta(2)-AR retained its ability to undergo ligand-induced conformational changes. The data from this study suggest that the immobilized beta(2)-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor.
Beigi
Farideh
F
Laboratory of Clinical Investigation and Laboratory of Cardiovascular Science, NIA, NIH, Baltimore, Maryland 21224-6825, USA.
Chakir
Khalid
K
Xiao
Rui-Ping
RP
Wainer
Irving W
IW
D000319:Adrenergic beta-Antagonists / Q000302:isolation & purification; D000818:Animals; D001665:Binding Sites; D002078:Butoxamine / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry; D051379:Mice; D009248:Nadolol / Q000302:isolation & purification; D011412:Propanolamines; D011433:Propranolol / Q000302:isolation & purification; D011487:Protein Conformation; D011941:Receptors, Adrenergic / Q000737:chemistry* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D000319:Adrenergic beta-Antagonists; D008024:Ligands; D008565:Membrane Proteins; D011412:Propanolamines; D011941:Receptors, Adrenergic; D043562:Receptors, G-Protein-Coupled; D002078:Butoxamine; D009248:Nadolol; D011433:Propranolol; C033394:CGP 12177