Probing human beta 1- and beta 2-adrenoceptors with domain-specific fusion protein antibodies

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 beta 1- or beta 2-adrenoceptors with...

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Bibliographic Details
Published in:European journal of pharmacology 1996-11, Vol.316 (1), p.111-121
Main Authors: Jahns, R, Siegmund, C, Jahns, V, Reiländer, H, Maidhof, A, Müller-Esterl, W, Lohse, M J, Boege, F
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antibody Specificity
Humans
Microscopy, Fluorescence
Precipitin Tests
Protein Structure, Secondary
Rabbits
Radioligand Assay
Receptors, Adrenergic, beta-1 - analysis
Receptors, Adrenergic, beta-2 - analysis
Recombinant Fusion Proteins - immunology
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Summary: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 beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-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 beta 1- or the beta 2-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.
ISSN:0014-2999