Probing human β1- and β2-adrenoceptors with domain-specific fusion protein antibodies

• Published in: European journal of pharmacology Vol. 316; no. 1; pp. 111 - 121
• Main Authors: Jahns, Roland, Siegmund, Christian, Jahns, Valérie, Reiländer, Helmut, Maidhof, Armin, Müller-Esterl, Werner, Lohse, Martin J., Boege, Fritz
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 28.11.1996; Elsevier
• Subjects: Antibody; Biological and medical sciences; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Fusion protein; Miscellaneous; Molecular and cellular biology; β1-Adrenoceptor; β2-Adrenoceptor; β 2-Adrenoceptor; Fusion protein; Antibody; β 1-Adrenoceptor; Human; Molecular structure; Escherichia coli; Molecular interaction; β2-Adrenergic receptor; Probe; In vitro; β1-Adrenergic receptor; Investigation method; Immunological investigation; Established cell line; Bacteria; Enterobacteriaceae
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/S0014-2999(96)00654-1

In order to generate antibodies suitable for immunological studies on β-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 β 1- or β 2-adrenoceptors with bacterial glutathione- S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human β 1- or β 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the β 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 β 1- or the β 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 β-adrenoceptors constitutively expressed in human A431 cells, where we observed a patched membrane distribution of the receptors.