Characterization of Nuclear Neurokinin 3 Receptor Expression in Rat Brain

Abstract Ligand-induced translocation of the G-protein-coupled receptor, neurokinin 3 (NK3-R), to the nucleus of hypothalamic neurons was reported using antibodies (ABs) raised against the C-terminal region of NK3-R. The current work was undertaken to substantiate the ability of NK3-R to enter the nucleus and identify which portion of the NK3-R molecule enters the nucleus. ABs directed at epitopes in the N-terminal and second extracellular loop of the rat NK3-R molecule were used to evaluate western blots of whole tissue homogenates and nuclear fractions from multiple brain areas. Specificity of the protein bands recognized by these ABs was demonstrated using Chinese hamster ovary (CHO) cells transfected with rat or human NK3-R. Both ABs prominently recognized a diffuse protein band of ∼56–65 kDa (56 kDa=predicted size) and distinct ∼70-kDa and 95-kDa proteins in homogenates of multiple brain areas. The ∼95-kDa protein recognized by the extracellular loop AB was enriched in nuclear fractions. Recognition of these proteins by ABs directed at different regions of the NK3-R supports their identification as NK3-R. The size differences reflect variable glycosylation and possibly linkage to different cytosolic and nuclear proteins. Recognition of protein bands by both ABs in nuclear fractions is consistent with the full-length NK3-R entering the nucleus. Hypotension increased the density of the ∼95-kDa band in nuclear fractions from the supraoptic nucleus indicating activity-induced nuclear translocation. Since NK3-R is widely distributed in the CNS, the presence of NK3-R in nuclei from multiple brain regions suggests that it may broadly influence CNS gene expression in a ligand-dependent manner.