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

SUMMARY

1. The differential identification of the angiotensin AT_1A and AT_1B receptor subtypes is impaired by the existing > 96% homology of both receptors. In the present study, we characterized two polyclonal rabbit peptide antibodies, namely α-AT_1A and α-AT_1B, that recognize the C-terminal region of mouse AT_1A and AT_1B 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 α-AT_1A antibody showed 2.5- to fourfold higher selectivity for its AT_1A receptor target peptide (1A-PEP) compared with the non-specific AT_1B receptor peptide (1B-PEP). In contrast, the α-AT_1B 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 α-AT_1B antibody, the α-AT_1A antibody selectively recognized the AT_1A 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 AT_1A and AT_1B receptors (α-AT_1A and α-AT_1B), of which the α-AT_1A antibody has the capability to distinguish AT_1A receptor types in immunological approaches.