Mutation of Asn293 to Asp in transmembrane helix VI abolishes agonist-induced
but not constitutive activity of the beta(2)-adrenergic receptor
A. Hannawacker, C. Krasel, and M. Lohse. Mol Pharmacol, 62 (6):
1431-7(December 2002)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..
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.
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.
%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
}