Functional characterization of a human receptor for neuropeptide FF and related peptides

1 Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-ba sed assay as screening tool, that an orphan GPCR, previously designated HLW AR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2 Binding experiments were performed with a new radioiodinated probe, [I-12 5]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chine se hamster ovary (CHO) cell membranes expressing NPFFR bound [I-125]-EYF wi th a K-d of 0.06 nM. Various NPFF analogues and related peptides inhibited [I-125]-EYF specific binding with the following rank order (K-i): human NPA F (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF ( 0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH2 (3.25 nM), FMRF- NH2 (10.5 nM) and NPSF (12.1 nM). 3 The stimulatory activity of the same set of peptides was measured by a fu nctional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequ orin. The rank order of potency was consistent with the results of the bind ing assay. 4 Membranes from NPFFR expressing CHO cells bound GTP gamma[S-35] in the pr esence of SQA-NPFF. This functional response was prevented by pertussis tox in treatment, demonstrating the involvement of Gi family members. 5 SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombina nt CHO cells in a dose dependent manner. This response was abolished as wel l by pertussis toxin pre-treatment. 6 RT-PCR analysis of human tissues mRNA revealed that expression of NPFFR w as mainly detected in placenta, thymus and at lower levels in pituitary gla nd, spleen and testis.