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 a gonists to beta(2)-adrenergic receptors rye used receptor models to id entify potential binding sites for the beta-OH-group of the ligand, wh ich 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 A la did not change the binding of either isoproterenol isomer as reveal ed after transient expression in human embryonic kidney (HEK)-293 cell s, In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Aden ylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoprot erenol, In a series of agonists the loss of affinity in the Leu-293 mu tant receptor was strongly correlated with the intrinsic activity of t he compounds, Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for bath receptors, Ste reospecific recognition of antagonists was unaltered in the Leu-293 mu tant receptor, These data indicate a relationship between stereospecif icity and intrinsic activity of agonists and suggest that Asn-293 is i mportant for both properties of the agonist-receptor interaction.