Article,

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

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Eur J Pharmacol, 316 (1): 111-21 (November 1996)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. 1996 Nov 28;316(1):111-21..

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 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)-5,7-3Hbenzimidazol-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 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.

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