Targeting human neuropeptide Y1 receptor (NPY1R) using a monoclonal antibody that targets an extracellular epitope.

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Background: Neuropeptide Y1 Receptor (NPY1R), a G protein-coupled receptor, is essential for regulating appetite, energy balance, and emotional responses, and is implicated in pathological disorders like obesity, anxiety and cancers. Currently, only small molecule reagents are available for assessing the expression of NPY1R in cancers. This study provides evidence supporting validation of one monoclonal antibody, mAb2A10, which binds an epitope in extracellular loop 2 of human NPY1R. Methods: The immediate aim was the validation of the monoclonal antibody mAb2A10 using a COS-7 cell line developed to overexpress human NPY1R (clone D9), and comparison with ER-positive MCF-7 breast cancer cells and Jurkat cells using FACS analysis, Western blotting, and confocal microscopy. The FLAG-NPY1R construct was introduced into COS-7 cells via plasmid transfection, followed by successive selection with puromycin to establish stable cell lines. Using flow cytometry, an anti-FLAG:568 antibody was used to identify a high NPY1R-expressing clone, D9. Results: Western blotting identified a ~136 kDa band in the high NPY1R-expressing D9 clone, MCF-7 and Jurkat cells), which was reduced when cells were grown in non-estrogenic media (charcoal stripped phenol red-free media plus FBS), indicating that the estrogenic effect regulates NPY1R at the protein level. The estrogen receptor (ER) exerts a positive effect on the activity of NPY1R; however, it is not yet established whether this affects mRNA expression or protein activation. Confocal microscopy supported these findings by confirming specific cellular localisation of NPY1R in D9 and MCF-7 cells. Conclusions: Therefore, the antibody validation process confirmed the specificity of mAb2A10 for FLAG-NPY1R. These findings collectively support the antibody's potential as a diagnostic or therapeutic tool for identifying NPY1R-expressing cancers including breast cancer.