Probing human beta1- and beta2 -adrenoceptors with domain-specific
fusion protein antibodies
R. Jahns, C. Siegmund, V. Jahns, H. Reilander, A. Maidhof, W. Muller-Esterl, M. Lohse, und F. Boege. Eur J Pharmacol, 334 (1):
115-26(September 1997)Jahns, R Siegmund, C Jahns, V Reilander, H Maidhof, A Muller-Esterl,
W Lohse, M J Boege, F Netherlands European journal of pharmacology
Eur J Pharmacol. 1997 Sep 3;334(1):115-26..
Zusammenfassung
In order to generate antibodies suitable for immunological studies
on beta-adrenoceptors constitutively expressed at low levels in cells
or tissues we have produced fusion proteins of the amino- and carboxy-terminus,
and the second extracellular loop of the human beta1- or beta2-adrenoceptors
with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies
raised against these fusion proteins strongly reacted with intact
human beta1- or beta2-adrenoceptors in a subtype- and domain-specific
manner. Antibodies directed against the second extracellular loop
of the beta1-adrenoceptor reacted stronger with non-denatured receptors
and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-5,7-3Hbenzimidazol-2-one
(3HCGP 12 177), indicating a specific interaction with the native
receptor. In contrast, antibodies directed against carboxy- and amino-terminal
receptor domains reacted strongly both with denatured and non-denatured
receptors but did not interfere with binding of 3HCGP 12 177. Affinity
purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor
subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent
assay, Western blotting, immunoprecipitation, and indirect immunofluorescence
microscopy. Moreover, we could demonstrate that avidity, titers,
and specificity of these antibodies were high enough for studying
beta-adrenoceptors constitutively expressed in human A431 cells,
where we observed a patched membrane distribution of the receptors.
Jahns, R Siegmund, C Jahns, V Reilander, H Maidhof, A Muller-Esterl,
W Lohse, M J Boege, F Netherlands European journal of pharmacology
Eur J Pharmacol. 1997 Sep 3;334(1):115-26.
%0 Journal Article
%1 Jahns1997
%A Jahns, R.
%A Siegmund, C.
%A Jahns, V.
%A Reilander, H.
%A Maidhof, A.
%A Muller-Esterl, W.
%A Lohse, M. J.
%A Boege, F.
%D 1997
%J Eur J Pharmacol
%K Animals Antibodies/*immunology Antibody Assay/methods Blotting, Enzyme-Linked Fluorescent Fusion Humans Immunosorbent Indirect Precipitin Proteins/chemistry/immunology Rabbits Recombinant Specificity Technique, Tests Western beta-1/chemistry/*immunology beta-2/chemistry/*immunology Receptor Adrenergic
%N 1
%P 115-26
%T Probing human beta1- and beta2 -adrenoceptors with domain-specific
fusion protein antibodies
%U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9346338
%V 334
%X In order to generate antibodies suitable for immunological studies
on beta-adrenoceptors constitutively expressed at low levels in cells
or tissues we have produced fusion proteins of the amino- and carboxy-terminus,
and the second extracellular loop of the human beta1- or beta2-adrenoceptors
with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies
raised against these fusion proteins strongly reacted with intact
human beta1- or beta2-adrenoceptors in a subtype- and domain-specific
manner. Antibodies directed against the second extracellular loop
of the beta1-adrenoceptor reacted stronger with non-denatured receptors
and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-5,7-3Hbenzimidazol-2-one
(3HCGP 12 177), indicating a specific interaction with the native
receptor. In contrast, antibodies directed against carboxy- and amino-terminal
receptor domains reacted strongly both with denatured and non-denatured
receptors but did not interfere with binding of 3HCGP 12 177. Affinity
purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor
subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent
assay, Western blotting, immunoprecipitation, and indirect immunofluorescence
microscopy. Moreover, we could demonstrate that avidity, titers,
and specificity of these antibodies were high enough for studying
beta-adrenoceptors constitutively expressed in human A431 cells,
where we observed a patched membrane distribution of the receptors.
@article{Jahns1997,
abstract = {In order to generate antibodies suitable for immunological studies
on beta-adrenoceptors constitutively expressed at low levels in cells
or tissues we have produced fusion proteins of the amino- and carboxy-terminus,
and the second extracellular loop of the human beta1- or beta2-adrenoceptors
with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies
raised against these fusion proteins strongly reacted with intact
human beta1- or beta2-adrenoceptors in a subtype- and domain-specific
manner. Antibodies directed against the second extracellular loop
of the beta1-adrenoceptor reacted stronger with non-denatured receptors
and decreased the affinity of the 3H-labelled antagonist (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one
([3H]CGP 12 177), indicating a specific interaction with the native
receptor. In contrast, antibodies directed against carboxy- and amino-terminal
receptor domains reacted strongly both with denatured and non-denatured
receptors but did not interfere with binding of [3H]CGP 12 177. Affinity
purified antibodies were used for detecting the beta1- or the beta2-adrenoceptor
subtype heterologously produced in Sf9 cells by enzyme-linked immunosorbent
assay, Western blotting, immunoprecipitation, and indirect immunofluorescence
microscopy. Moreover, we could demonstrate that avidity, titers,
and specificity of these antibodies were high enough for studying
beta-adrenoceptors constitutively expressed in human A431 cells,
where we observed a patched membrane distribution of the receptors.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Jahns, R. and Siegmund, C. and Jahns, V. and Reilander, H. and Maidhof, A. and Muller-Esterl, W. and Lohse, M. J. and Boege, F.},
biburl = {https://www.bibsonomy.org/bibtex/291da54a2eca2a106ceeebbb6acb819b8/pharmawuerz},
endnotereftype = {Journal Article},
interhash = {35a416a790f596e5507a50913b39f16b},
intrahash = {91da54a2eca2a106ceeebbb6acb819b8},
issn = {0014-2999 (Print) 0014-2999 (Linking)},
journal = {Eur J Pharmacol},
keywords = {Animals Antibodies/*immunology Antibody Assay/methods Blotting, Enzyme-Linked Fluorescent Fusion Humans Immunosorbent Indirect Precipitin Proteins/chemistry/immunology Rabbits Recombinant Specificity Technique, Tests Western beta-1/chemistry/*immunology beta-2/chemistry/*immunology Receptor Adrenergic},
month = {Sep 3},
note = {Jahns, R Siegmund, C Jahns, V Reilander, H Maidhof, A Muller-Esterl,
W Lohse, M J Boege, F Netherlands European journal of pharmacology
Eur J Pharmacol. 1997 Sep 3;334(1):115-26.},
number = 1,
pages = {115-26},
shorttitle = {Probing human beta1- and beta2 -adrenoceptors with domain-specific
fusion protein antibodies},
timestamp = {2010-12-14T18:22:40.000+0100},
title = {Probing human beta1- and beta2 -adrenoceptors with domain-specific
fusion protein antibodies},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9346338},
volume = 334,
year = 1997
}