Neurokinin 3 receptor (NK3R) immunoreactivity (IR) in supraoptic nucleus (SON): Impact of antigen epitope on immunohistochemical (IHC) localization

Abstract

Activation of NK3R results in receptor internalization and nuclear localization of NK3R‐IR in SON when a C‐terminal directed antibody (Ab; aa 438‐452; Novus) is used (Howe et al, J. Neurosci. 24:10103, 04). To further characterize this IR, we generated 2 Abs against each of 3 additional epitopes in the NK3R (N‐terminal aa 6‐18; C‐terminal aa 376‐396 and aa 408‐420). Western blot (WB) and IHC with confocal microscopy were used to characterize the IR. On WB, all Abs recognized ~70kD protein in microdissected rat SON and in CHO cells transfected with rat NK3R. This band was absent in non‐transfected CHO cells and following peptide preabsorption of the Ab. The larger than predicted size (e.g. ~54kD) probably reflects post‐translational modification of the receptor. IHC with the N‐terminal Abs duplicated the membrane labeling seen with the Novus Ab, but prominent membrane IR was not evident with the other Abs and none of the new Abs replicated the hypotension induced punctate nuclear staining evident with the Novus Ab. These observations were not altered by modification of the fixation protocol nor Ab dilution. These findings demonstrate that Abs generated against multiple epitopes of the NK3R recognize a similar protein on WB. In contrast, the epitope target of NK3R Ab alters IHC detected cellular localization of the receptor perhaps due to differential Ab access to the epitope in the intact cell. Supported by NIH R21‐NS059569.