%0 Journal Article %1 Hannawacker2002 %A Hannawacker, A. %A Krasel, C. %A Lohse, M. J. %D 2002 %J Mol Pharmacol %K *Mutation Acid Acid/genetics Adrenergic Agonists/pharmacology Amino Animals Asparagine/genetics Aspartic CHO Cricetinae Membrane Protein Proteins/chemistry/genetics/*metabolism Secondary Structure, Substitution Tertiary beta-2/agonists/chemistry/genetics/*metabolism Receptor Cell %N 6 %P 1431-7 %T Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the beta(2)-adrenergic receptor %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12435811 %V 62 %X 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.

@article{Hannawacker2002, abstract = {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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Hannawacker, A. and Krasel, C. and Lohse, M. J.}, biburl = {https://www.bibsonomy.org/bibtex/237112c2c4f077eaa3a25bf81b30ea0d4/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {3a0b9e22179c04cb309bf49810656aef}, intrahash = {37112c2c4f077eaa3a25bf81b30ea0d4}, issn = {0026-895X (Print) 0026-895X (Linking)}, journal = {Mol Pharmacol}, keywords = {*Mutation Acid Acid/genetics Adrenergic Agonists/pharmacology Amino Animals Asparagine/genetics Aspartic CHO Cricetinae Membrane Protein Proteins/chemistry/genetics/*metabolism Secondary Structure, Substitution Tertiary beta-2/agonists/chemistry/genetics/*metabolism Receptor Cell}, month = Dec, note = {Hannawacker, Annette Krasel, Cornelius Lohse, Martin J Research Support, Non-U.S. Gov't United States Molecular pharmacology Mol Pharmacol. 2002 Dec;62(6):1431-7.}, number = 6, pages = {1431-7}, shorttitle = {Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the beta(2)-adrenergic receptor}, timestamp = {2010-12-14T18:22:40.000+0100}, title = {Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced but not constitutive activity of the beta(2)-adrenergic receptor}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12435811}, volume = 62, year = 2002 }