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)-5,7-3Hbenzimidazol-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.
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