A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

• Published in: Analytical biochemistry Vol. 453; pp. 50 - 54
• Main Authors: Bray, Lauriane, Moulédous, Lionel, Tafani, Jean A.M., Germanier, Maryse, Zajac, Jean-Marie
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.05.2014
• Subjects: Affinity labeling; Affinity Labels - chemical synthesis; Affinity Labels - chemistry; Amino Acid Sequence; Animals; Humans; Iodine Radioisotopes; Ligands; Mice; Mice, Inbred C57BL; Molecular Weight; Neuroblastoma - metabolism; Neuropeptide FF; Olfactory Bulb - metabolism; Oligopeptides - analysis; Oligopeptides - chemistry; Phenylalanine - analogs & derivatives; Phenylalanine - chemistry; Photolysis; Radioiodination; Radioligand Assay - methods; Rats; Receptor; Receptors, Neuropeptide - analysis; Receptors, Neuropeptide - chemistry; Receptors, Neuropeptide - metabolism; Tumor Cells, Cultured; Radioiodination; Neuropeptide FF; Receptor; Affinity labeling
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2014.02.029

A new radioiodinated photoaffinity compound, [125I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [125I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [125I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.