Identifying new peptides with enhanced binding properties to the Angiotensin II type 2 receptor (AT2R) can lead to the developing of novel antifibrotic agents [1]. While unabundant in most adult tissues, AT2R can be markedly upregulated in cancer, where its functioning remains elusive [2]. AT2R is largely unexplored, and previous studies have shown that commercially available AT2R antibodies lack selectivity and exhibit markedly different immunocytochemical reactivity patterns with multiple off-target interactions, thus making it challenging to visualise AT2R. The lack of selective antibodies for AT2R has hindered research in the AT2R field [3].
To solve this problem, here we present a new strategy utilising Angiotensin III -based peptides as new tools to examine AT2R. In the present study, we developed novel highly selective AT2R peptides to detect and visualise AT2R in stable over-expressing cells, native cells and animal tissues in health and disease. Peptides contain an electrophilic or photoreactive moiety capable of covalent attachment to the receptor and a fluorophore or biotin for visualisation. Despite the additional modifications, the radioligand binding assays demonstrated excellent selectivity for several peptide ligands over the closest receptor target, Angiotensin II type 1 receptor [4]. We also proved the absence of potential off-target interactions with other Renin-Angiotensin system components. We confirmed the selectivity of the novel peptide affinity labels by utilising a selective AT2R antagonist, PD123319, and mouse AT2R KO tissues. High AT2R expression levels were detected in WT mouse tissues in the kidney, adrenal gland, brain, and multiple cancer cell lines using fluorescence microscopy methods. We also observed AT2R expression changes in various disease models using these peptides. This novel approach of utilising AT2R-selective peptide affinity labels provides further insight into AT2R localisation and function in health and disease.