Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.
To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.
Wieland
K
K
Department of Pharmacology, University of Würzburg, Germany.
Zuurmond
H M
HM
Krasel
C
C
Ijzerman
A P
AP
Lohse
M J
MJ
D000262:Adenylyl Cyclases / Q000187:drug effects; D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000378:metabolism*; D000526:Alprenolol / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D003198:Computer Simulation; D006224:Cricetinae; D006801:Humans; D007545:Isoproterenol / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008790:Metoprolol / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011433:Propranolol / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013237:Stereoisomerism
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D012694:Serine; D001216:Asparagine; D000526:Alprenolol; D011433:Propranolol; D000262:Adenylyl Cyclases; D008790:Metoprolol; D007545:Isoproterenol