Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled
receptors
P. Sohlemann, M. Hekman, M. Puzicha, C. Buchen, and M. Lohse. Eur J Biochem, 232 (2):
464-72(September 1995)Sohlemann, P Hekman, M Puzicha, M Buchen, C Lohse, M J Research Support,
Non-U.S. Gov't Germany European journal of biochemistry / FEBS Eur
J Biochem. 1995 Sep 1;232(2):464-72..
Abstract
beta-arrestin is a cytosolic protein thought to be responsible for
uncoupling agonist-activated beta 2-adrenergic receptors from their
guanine-nucleotide-binding proteins (G-protein) subsequent to receptor
phosphorylation by the beta-adrenergic receptor kinase (beta ARK).
In order to investigate this interaction, we generated a recombinant
baculovirus for the expression of beta-arrestin in Sf9 insect cells.
Apparently homogeneous beta-arrestin preparations were obtained in
a one-step purification on heparin-Sepharose. Purified beta-arrestin
bound to rhodopsin in a phosphorylation-dependent plus light-dependent
manner. Binding to beta 2-adrenergic receptors was investigated using
purified receptors reconstituted into lipid vesicles. The accessibility
of the reconstituted receptors was determined using the agonist isoproterenol
for the ligand-binding site and an antibody binding to an attached
myc tag for the C-terminus, the site of receptor phosphorylation.
On the basis of these data, the binding of purified beta-arrestin
to beta ARK-phosphorylated beta 2-adrenergic receptors was found
to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor.
beta-arrestin also bound to receptors which had been completely dephosphorylated
with acid phosphatase, but the affinity was approximately 30-fold
lower. In contrast to regulation by phosphorylation, binding of agonists
or antagonists to the receptors had negligible effects on beta-arrestin
binding. Finally, beta-arrestin and beta ARK were shown to be capable
of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated
adenylyl cyclase activity of cell membranes. These data show that
high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic
receptors occurs in a beta ARK-dependent manner and is sufficient
to impair adenylyl cyclase stimulation by the receptors.
Sohlemann, P Hekman, M Puzicha, M Buchen, C Lohse, M J Research Support,
Non-U.S. Gov't Germany European journal of biochemistry / FEBS Eur
J Biochem. 1995 Sep 1;232(2):464-72.
%0 Journal Article
%1 Sohlemann1995
%A Sohlemann, P.
%A Hekman, M.
%A Puzicha, M.
%A Buchen, C.
%A Lohse, M. J.
%D 1995
%J Eur J Biochem
%K *Arrestins AMP-Dependent Adenylate Animals Antigens/genetics/*metabolism Binding Cell Cyclase/metabolism Cyclic Expression Eye GTP-Binding Gene Humans Kinases Kinases/metabolism Kinetics Line Outer Phosphorylation Protein Proteins/genetics/*metabolism Proteins/genetics/metabolism Receptor Recombinant Rhodopsin/metabolism Rod Segment/metabolism Spodoptera beta-2/chemistry/genetics/*metabolism beta-Adrenergic Proteins/metabolism Adrenergic
%N 2
%P 464-72
%T Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled
receptors
%U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7556195
%V 232
%X beta-arrestin is a cytosolic protein thought to be responsible for
uncoupling agonist-activated beta 2-adrenergic receptors from their
guanine-nucleotide-binding proteins (G-protein) subsequent to receptor
phosphorylation by the beta-adrenergic receptor kinase (beta ARK).
In order to investigate this interaction, we generated a recombinant
baculovirus for the expression of beta-arrestin in Sf9 insect cells.
Apparently homogeneous beta-arrestin preparations were obtained in
a one-step purification on heparin-Sepharose. Purified beta-arrestin
bound to rhodopsin in a phosphorylation-dependent plus light-dependent
manner. Binding to beta 2-adrenergic receptors was investigated using
purified receptors reconstituted into lipid vesicles. The accessibility
of the reconstituted receptors was determined using the agonist isoproterenol
for the ligand-binding site and an antibody binding to an attached
myc tag for the C-terminus, the site of receptor phosphorylation.
On the basis of these data, the binding of purified beta-arrestin
to beta ARK-phosphorylated beta 2-adrenergic receptors was found
to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor.
beta-arrestin also bound to receptors which had been completely dephosphorylated
with acid phosphatase, but the affinity was approximately 30-fold
lower. In contrast to regulation by phosphorylation, binding of agonists
or antagonists to the receptors had negligible effects on beta-arrestin
binding. Finally, beta-arrestin and beta ARK were shown to be capable
of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated
adenylyl cyclase activity of cell membranes. These data show that
high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic
receptors occurs in a beta ARK-dependent manner and is sufficient
to impair adenylyl cyclase stimulation by the receptors.
@article{Sohlemann1995,
abstract = {beta-arrestin is a cytosolic protein thought to be responsible for
uncoupling agonist-activated beta 2-adrenergic receptors from their
guanine-nucleotide-binding proteins (G-protein) subsequent to receptor
phosphorylation by the beta-adrenergic receptor kinase (beta ARK).
In order to investigate this interaction, we generated a recombinant
baculovirus for the expression of beta-arrestin in Sf9 insect cells.
Apparently homogeneous beta-arrestin preparations were obtained in
a one-step purification on heparin-Sepharose. Purified beta-arrestin
bound to rhodopsin in a phosphorylation-dependent plus light-dependent
manner. Binding to beta 2-adrenergic receptors was investigated using
purified receptors reconstituted into lipid vesicles. The accessibility
of the reconstituted receptors was determined using the agonist isoproterenol
for the ligand-binding site and an antibody binding to an attached
myc tag for the C-terminus, the site of receptor phosphorylation.
On the basis of these data, the binding of purified beta-arrestin
to beta ARK-phosphorylated beta 2-adrenergic receptors was found
to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor.
beta-arrestin also bound to receptors which had been completely dephosphorylated
with acid phosphatase, but the affinity was approximately 30-fold
lower. In contrast to regulation by phosphorylation, binding of agonists
or antagonists to the receptors had negligible effects on beta-arrestin
binding. Finally, beta-arrestin and beta ARK were shown to be capable
of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated
adenylyl cyclase activity of cell membranes. These data show that
high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic
receptors occurs in a beta ARK-dependent manner and is sufficient
to impair adenylyl cyclase stimulation by the receptors.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Sohlemann, P. and Hekman, M. and Puzicha, M. and Buchen, C. and Lohse, M. J.},
biburl = {https://www.bibsonomy.org/bibtex/27b537bf6cde44158ccbc56dd59a49b24/pharmawuerz},
endnotereftype = {Journal Article},
interhash = {245e5223aa110c01af5ac9adde691a53},
intrahash = {7b537bf6cde44158ccbc56dd59a49b24},
issn = {0014-2956 (Print) 0014-2956 (Linking)},
journal = {Eur J Biochem},
keywords = {*Arrestins AMP-Dependent Adenylate Animals Antigens/genetics/*metabolism Binding Cell Cyclase/metabolism Cyclic Expression Eye GTP-Binding Gene Humans Kinases Kinases/metabolism Kinetics Line Outer Phosphorylation Protein Proteins/genetics/*metabolism Proteins/genetics/metabolism Receptor Recombinant Rhodopsin/metabolism Rod Segment/metabolism Spodoptera beta-2/chemistry/genetics/*metabolism beta-Adrenergic Proteins/metabolism Adrenergic},
month = {Sep 1},
note = {Sohlemann, P Hekman, M Puzicha, M Buchen, C Lohse, M J Research Support,
Non-U.S. Gov't Germany European journal of biochemistry / FEBS Eur
J Biochem. 1995 Sep 1;232(2):464-72.},
number = 2,
pages = {464-72},
shorttitle = {Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled
receptors},
timestamp = {2010-12-14T18:22:42.000+0100},
title = {Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled
receptors},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7556195},
volume = 232,
year = 1995
}