Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the beta(2)-adrenergic receptor.

The beta(2)-adrenergic receptor has been shown to display significant constitutive activity (i.e., in the absence of agonist) in addition to agonist-induced activation. Various studies have suggested that a movement in transmembrane helix VI plays a role in activation of various G-protein-coupled receptors. Here we show that a mutation in this domain of the beta(2)-adrenergic receptor abolishes agonist activation but not constitutive activity. An Asn293Asp mutant of the human beta(2)-adrenergic receptor was expressed either transiently in COS-7 cells or stably in Chinese hamster ovary cells. The mutant receptors were unable to couple to G(s), as seen by the lack of high-affinity agonist binding as well as a reduction of the affinities of several agonists correlating with their intrinsic activities. The mutant receptors caused only minimal activation of adenylyl cyclase (2.5% of wild-type activity) and also failed to show agonist-induced phosphorylation by G-protein-coupled receptor kinase 2. In contrast, the mutant receptors were much less affected in their constitutive activity: transient transfection of wild-type and mutant receptors into COS-7 cells caused an increase in intracellular cAMP-levels that was dependent on the level of receptor expression and was maximally 5.4-fold for the mutant and 6.8-fold for the wild-type receptors (67% of wild-type activity). Introduction of the Asn293Asp mutation into a constitutively active mutant receptor did not affect the constitutive activity of this mutant. These results underscore the importance of transmembrane helix VI in controlling agonist-induced activation of the receptor and suggest that constitutive activity is different from agonist-induced activity. Furthermore, they indicate that Asn293 is a key residue in transferring conformational information from the agonist-binding site to the intracellular surface.