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Journal Article

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


Reiländer,  Helmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Jahns, R., Siegmund, C., Jahns, V., Reiländer, H., Maidhof, A., Müller-Esterl, W., et al. (1996). Probing human β1- and β2-adrenoceptors with domain-specific fusion protein antibodies. European Journal of Pharmacology, 316(1), 111-121. doi:10.1016/S0014-2999(96)00654-1.

Cite as: http://hdl.handle.net/21.11116/0000-0007-133A-3
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 β1- or β2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human β1- or β2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the β1-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 β1- or the β2-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.