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A new radioiodinated photoaffinity compound, [ 125 I]YE(Bpa)WSLAAPQRFNH 2 , 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 NPFF 2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction ( K D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF 2 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 [ 125 I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF 2 receptor with apparent molecular weights of 140 and 95 kDa 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 NPFF 2 receptor is observed. [ 125 I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF 2 receptors in vitro.

Neuropeptide FF (NPFF, FLFQPQRFamide) receptors modulate endogenous opioid functions. Here, we report the solubilization of the human NPFF2 receptor expressed in Chinese hamster ovary (CHO) cells by the zwitterionic detergent Chaps. Chaps solubilization resulted in the abolishment of specific agonist binding activity, which was restored by a polyethylene glycol (PEG) precipitation method. Reincorporation after the precipitation step into liposomes made of endogenous lipids issued from CHO membranes or exogenous lipids significantly enhanced the specific agonist binding activity and G-protein coupling. This method of solubilization and lipid reconstitution could be useful for studies of NPFF receptors.

Functional and structural studies on G-protein-coupled receptors (GPCRs) at molecular levels require producing and purifying high levels of receptors, and recombinant mammalian cell expression systems constitute the best systems to obtain receptors resembling those expressed in natural environments. In the course of increasing GPCR expression in Chinese hamster ovary (CHO) cells, we have expressed mu (μ)- and kappa (κ)-opioid receptors and neuropeptide FF 1 and FF 2 receptors (NPFF 1 and NPFF 2 , respectively) in dimethyl sulfoxide. This treatment did not modify the affinity ( K d ) for any receptor, but a significant increase in functional expression levels was observed for all receptors with the noticeable exception of NPFF 1 .