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 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.
Jahns
R
R
Department of Pharmacology, University of Würzburg, Germany. jahns@wpxx02.toxi.uni-wuerzburg.de
Siegmund
C
C
Jahns
V
V
Reiländer
H
H
Maidhof
A
A
Müller-Esterl
W
W
Lohse
M J
MJ
Boege
F
F
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D006801:Humans; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D011817:Rabbits; D011869:Radioligand Assay; D018342:Receptors, Adrenergic, beta-1 / Q000032:analysis*; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D000906:Antibodies; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins