Ligand-induced phosphorylation/dephosphorylation of the endogenous
bradykinin B2 receptor from human fibroblasts
A. Blaukat, S. Alla, M. Lohse, und W. Muller-Esterl. J Biol Chem, 271 (50):
32366-74(Dezember 1996)Blaukat, A Alla, S A Lohse, M J Muller-Esterl, W Research Support,
Non-U.S. Gov't United states The Journal of biological chemistry
J Biol Chem. 1996 Dec 13;271(50):32366-74..
Zusammenfassung
We have studied the ligand-induced phosphorylation/dephosphorylation
of the bradykinin B2 receptor endogenously expressed in human HF-15
fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36),
derived from the extreme carboxyl terminus of the human B2 receptor,
precipitated the receptor from solubilized membranes of HF-15 cells
that had been labeled with 32Porthophosphate. A low basal level
of B2 receptor phosphorylation was found in the absence of a ligand.
Stimulation of the cells with the B2 receptor agonists bradykinin,
Lys0,Hyp3bradykinin, kallidin, and T-kinin resulted in a rapid
and efficient phosphorylation of the receptor. The B2 receptor antagonist
HOE140 and the B1 receptor agonist des-Arg9-bradykinin failed to
induce significant phosphorylation of the B2 receptor. Phosphoamino
acid analysis revealed that the B2 receptor is phosphorylated on
serine and threonine, but not on tyrosine residues. The ligand-induced
phosphorylation of the receptor was concentration-dependent, with
an apparent EC50 of 33 nM, and peaked at 1 min after challenge. The
kinin-stimulated phosphorylation of the B2 receptor was rapid and
transient and paralleled the kinetics of desensitization/resensitization
of the receptor as followed by Ca2+i release and radioligand binding
assay, respectively. The ligand-induced phosphorylation of the B2
receptor was independent of the protein kinase C pathway. In vitro
experiments suggest betaARK1 (beta-adrenergic receptor kinase) as
a candidate kinase that could mediate the homologous B2 receptor
phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively
blocked the dephosphorylation, but did not affect the internalization
of the B2 receptor, whereas inhibitors of receptor internalization
delayed its dephosphorylation. These finding point to a role of ligand-induced
phosphorylation in the desensitization and redistribution of the
bradykinin receptor in human fibroblasts.
Blaukat, A Alla, S A Lohse, M J Muller-Esterl, W Research Support,
Non-U.S. Gov't United states The Journal of biological chemistry
J Biol Chem. 1996 Dec 13;271(50):32366-74.
%0 Journal Article
%1 Blaukat1996
%A Blaukat, A.
%A Alla, S. A.
%A Lohse, M. J.
%A Muller-Esterl, W.
%D 1996
%J J Biol Chem
%K A/pharmacology Animals B2 Bradykinin Bradykinin/*metabolism Bradykinin/metabolism COS Cell Concanavalin Electrophoresis, Fibroblasts/metabolism Gel Humans Hydrolases/metabolism Line Monoester Phosphoric Phosphorylation Polyacrylamide Receptor
%N 50
%P 32366-74
%T Ligand-induced phosphorylation/dephosphorylation of the endogenous
bradykinin B2 receptor from human fibroblasts
%U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8943300
%V 271
%X We have studied the ligand-induced phosphorylation/dephosphorylation
of the bradykinin B2 receptor endogenously expressed in human HF-15
fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36),
derived from the extreme carboxyl terminus of the human B2 receptor,
precipitated the receptor from solubilized membranes of HF-15 cells
that had been labeled with 32Porthophosphate. A low basal level
of B2 receptor phosphorylation was found in the absence of a ligand.
Stimulation of the cells with the B2 receptor agonists bradykinin,
Lys0,Hyp3bradykinin, kallidin, and T-kinin resulted in a rapid
and efficient phosphorylation of the receptor. The B2 receptor antagonist
HOE140 and the B1 receptor agonist des-Arg9-bradykinin failed to
induce significant phosphorylation of the B2 receptor. Phosphoamino
acid analysis revealed that the B2 receptor is phosphorylated on
serine and threonine, but not on tyrosine residues. The ligand-induced
phosphorylation of the receptor was concentration-dependent, with
an apparent EC50 of 33 nM, and peaked at 1 min after challenge. The
kinin-stimulated phosphorylation of the B2 receptor was rapid and
transient and paralleled the kinetics of desensitization/resensitization
of the receptor as followed by Ca2+i release and radioligand binding
assay, respectively. The ligand-induced phosphorylation of the B2
receptor was independent of the protein kinase C pathway. In vitro
experiments suggest betaARK1 (beta-adrenergic receptor kinase) as
a candidate kinase that could mediate the homologous B2 receptor
phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively
blocked the dephosphorylation, but did not affect the internalization
of the B2 receptor, whereas inhibitors of receptor internalization
delayed its dephosphorylation. These finding point to a role of ligand-induced
phosphorylation in the desensitization and redistribution of the
bradykinin receptor in human fibroblasts.
@article{Blaukat1996,
abstract = {We have studied the ligand-induced phosphorylation/dephosphorylation
of the bradykinin B2 receptor endogenously expressed in human HF-15
fibroblasts. An antiserum (AS346) to a synthetic peptide (CRS36),
derived from the extreme carboxyl terminus of the human B2 receptor,
precipitated the receptor from solubilized membranes of HF-15 cells
that had been labeled with [32P]orthophosphate. A low basal level
of B2 receptor phosphorylation was found in the absence of a ligand.
Stimulation of the cells with the B2 receptor agonists bradykinin,
[Lys0,Hyp3]bradykinin, kallidin, and T-kinin resulted in a rapid
and efficient phosphorylation of the receptor. The B2 receptor antagonist
HOE140 and the B1 receptor agonist des-Arg9-bradykinin failed to
induce significant phosphorylation of the B2 receptor. Phosphoamino
acid analysis revealed that the B2 receptor is phosphorylated on
serine and threonine, but not on tyrosine residues. The ligand-induced
phosphorylation of the receptor was concentration-dependent, with
an apparent EC50 of 33 nM, and peaked at 1 min after challenge. The
kinin-stimulated phosphorylation of the B2 receptor was rapid and
transient and paralleled the kinetics of desensitization/resensitization
of the receptor as followed by [Ca2+]i release and radioligand binding
assay, respectively. The ligand-induced phosphorylation of the B2
receptor was independent of the protein kinase C pathway. In vitro
experiments suggest betaARK1 (beta-adrenergic receptor kinase) as
a candidate kinase that could mediate the homologous B2 receptor
phosphorylation. Inhibitors of protein phosphatases 1 and 2A effectively
blocked the dephosphorylation, but did not affect the internalization
of the B2 receptor, whereas inhibitors of receptor internalization
delayed its dephosphorylation. These finding point to a role of ligand-induced
phosphorylation in the desensitization and redistribution of the
bradykinin receptor in human fibroblasts.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Blaukat, A. and Alla, S. A. and Lohse, M. J. and Muller-Esterl, W.},
biburl = {https://www.bibsonomy.org/bibtex/25a1ee6d41221a157202c48191a55d5ca/pharmawuerz},
endnotereftype = {Journal Article},
interhash = {d2dd99cc923a78b7228af4978afa8819},
intrahash = {5a1ee6d41221a157202c48191a55d5ca},
issn = {0021-9258 (Print) 0021-9258 (Linking)},
journal = {J Biol Chem},
keywords = {A/pharmacology Animals B2 Bradykinin Bradykinin/*metabolism Bradykinin/metabolism COS Cell Concanavalin Electrophoresis, Fibroblasts/metabolism Gel Humans Hydrolases/metabolism Line Monoester Phosphoric Phosphorylation Polyacrylamide Receptor},
month = {Dec 13},
note = {Blaukat, A Alla, S A Lohse, M J Muller-Esterl, W Research Support,
Non-U.S. Gov't United states The Journal of biological chemistry
J Biol Chem. 1996 Dec 13;271(50):32366-74.},
number = 50,
pages = {32366-74},
shorttitle = {Ligand-induced phosphorylation/dephosphorylation of the endogenous
bradykinin B2 receptor from human fibroblasts},
timestamp = {2010-12-14T18:20:38.000+0100},
title = {Ligand-induced phosphorylation/dephosphorylation of the endogenous
bradykinin B2 receptor from human fibroblasts},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8943300},
volume = 271,
year = 1996
}