Isolation and identification of endogenous RFRP1 and RFRP3 in the mouse hypothalamus

Abstract

Although RFamide‐related peptide (RFRP) preproprotein sequence is known in mice, until now the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using immunoaffinity column conjugated with specific antibodies against the mouse C‐terminus of RFRP‐1 and RFRP‐3. Employing liquid chromatography coupled with mass spectrometry (LC‐MS) we demonstrated, that RFRP1 consists of 15 and RFRP3 of 10 amino acid residues (ANKVPHSAANLPLRF‐NH₂ and SHFPSLPQRF‐NH₂, respectively). To investigate the distribution of RFRPs in the mouse central nervous system (CNS), we performed immunohistochemical (IHC) staining of the brain sections collected from wild‐type (WT) and Rfrp knock‐out (Rfrp KO) animals. This data together with gene expression in multiple tissues provides strong confidence that RFRP‐immunoreactive (RFRP‐ir) neuronal cells are localized in the dorsomedial (DMH) and between DMH and the ventromedial (VMH) hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and the IHC visualization of targeting RFRPs neurons in the mice brain provide the basis for the investigations into a functional biology of RFRPs.

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