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

• Published in: British journal of pharmacology Vol. 133; no. 1; pp. 138 - 144
• Main Authors: Kotani, Masato, Mollereau, Catherine, Detheux, Michel, Le Poul, Emmanuel, Brézillon, Stéphane, Vakili, Jalal, Mazarguil, Honoré, Vassart, Gilbert, Zajac, Jean‐Marie, Parmentier, Marc
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-05-2001; Nature Publishing
• Subjects: Aequorin; aequorin assay; Animals; Binding, Competitive; Biological and medical sciences; Calcium - metabolism; Cell receptors; Cell structures and functions; CHO Cells; Cloning, Molecular; Colforsin - antagonists & inhibitors; Colforsin - pharmacology; Cricetinae; Cyclic AMP - metabolism; Fundamental and applied biological sciences. Psychology; G protein‐coupled receptor; Gene Expression Profiling; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Humans; Molecular and cellular biology; neuropeptide AF; Neuropeptide FF; Neuropeptide receptors; Oligopeptides - metabolism; orphan receptor; Pertussis Toxin; Receptors, Neuropeptide - agonists; Receptors, Neuropeptide - genetics; Receptors, Neuropeptide - metabolism; RNA, Messenger - analysis; RNA, Messenger - genetics; Substrate Specificity; Thermodynamics; Virulence Factors, Bordetella - pharmacology; Human; Rat; Rodentia; Animal origin; Tissue specificity; Tolerance; Opiates; Gene expression; In vitro; CHO cell line; Vertebrata; Mammalia; Pain; Established cell line; Peptidergic receptor; Recombinant protein; Neuropeptide FF; Orphan receptor; G protein
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0704038

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‐based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR.
Binding experiments were performed with a new radioiodinated probe, [125I]‐EYF, derived from the EFW‐NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [125I]‐EYF with a Kd of 0.06 nM. Various NPFF analogues and related peptides inhibited [125I]‐EYF specific binding with the following rank order (Ki): human NPAF (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).
The stimulatory activity of the same set of peptides was measured by a functional assay based on the co‐expression of NPFFR, Gα16 and apoaequorin. The rank order of potency was consistent with the results of the binding assay.
Membranes from NPFFR expressing CHO cells bound GTPγ[35S] in the presence of SQA‐NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of Gi family members.
SQA‐NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre‐treatment.
RT – PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.
British Journal of Pharmacology (2001) 133, 138–144; doi:10.1038/sj.bjp.0704038