Characterization of two polyclonal peptide antibodies that recognize the carboxy terminus of angiotensin II AT1A and AT1B receptors

Abstract

1. The differential identification of the angiotensin AT1A and AT1B receptor subtypes is impaired by the existing>96% homology of both receptors. In the present study, we characterized two polyclonal rabbit peptide antibodies, namely alpha-AT1A and alpha-AT1B, that recognize the C-terminal region of mouse AT1A and AT1B receptors, respectively. 2. In immunoblotting, both antibodies detected two major AT1 receptor-specific bands at sizes of 72.5 and 87.6 kDa in mouse tissues and in Neuro-2a cell lysates. In immunohistochemistry, antibodies demonstrated AT1 receptor-specific staining in renal proximal and distal tubules, as well as in kidney glomeruli. In addition, both antibodies stained AT1 receptors in Neuro-2a cells with G-protein receptor typical distribution. Dot-blot and ELISA analysis of the alpha-AT1A antibody showed 2.5- to fourfold higher selectivity for its AT1A receptor target peptide (1A-PEP) compared with the non-specific AT1B receptor peptide (1B-PEP). In contrast, the alpha-AT1B antibody showed high binding affinity towards its target peptide 1B-PEP, but also demonstrated high cross-reactivity for the non-specific peptide 1A-PEP (1.4- to twofold in ELISA and dot-blot analysis). In contrast with the lack of recognition by the alpha-AT1B antibody, the alpha-AT1A antibody selectively recognized the AT1A receptor fused to red fluorescence protein in transiently transfected Chinese hamster ovary cells. 3. In summary, we have generated two new peptide antibodies to the mouse AT1A and AT1B receptors (alpha-AT1A and alpha-AT1B), of which the alpha-AT1A antibody has the capability to distinguish AT1A receptor types in immunological approaches.