Characterization of a panel of six β(2)-adrenergic receptor antibodies by indirect immunofluorescence microscopy

BACKGROUND: The β(2)-adrenergic receptor (β(2)AR) is a primary target for medications used to treat asthma. Due to the low abundance of β(2)AR, very few studies have reported its localization in tissues. However, the intracellular location of β(2)AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β(2)AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β(2)AR in primary cell cultures. METHODS: A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β(2)AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β(2)AR were identified and used to localize native β(2)AR in primary cultures of rat airway smooth muscle and epithelial cells. β(2)AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol (([3H]DHA)). RESULTS: Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β(2)AR (Ab-Bethyl) specifically recognized human but not rat β(2)AR. An antibody developed against the C-terminal domain of the mouse β(2)AR (Ab-sc570) specifically recognized rat but not human β(2)AR. An antibody developed against 78 amino acids of the C-terminus of the human β(2)AR (Ab-13989) was capable of recognizing both rat and human β(2)ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β(2)AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface. CONCLUSION: Antibodies have been identified that recognize human β(2)AR, rat β(2)AR or both rat and human β(2)AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β(2)AR in tissues.