Structure–Activity Studies of RFamide Peptides Reveal Subtype‐Selective Activation of Neuropeptide FF1 and FF2 Receptors

Abstract

Selectivity is a major issue in closely related multiligand/multireceptor systems. In this study we investigated the RFamide systems of hNPFF₁R and hNPFF₂R that bind the endogenous peptide hormones NPFF, NPAF, NPVF, and NPSF. By use of a systematic approach, we characterized the role of the C-terminal dipeptide with respect to agonistic properties using synthesized [Xaa 7]NPFF and [Xaa 8]NPFF analogues. We were able to identify only slight differences in potency upon changing the position of Arg 7, as all modifications resulted in identical behavior at the NPFF₁R and NPFF₂R. However, the C-terminal Phe 8 was able to be replaced by Trp or His with only a minor loss in potency at the NPFF₂R relative to the NPFF₁R. Analogues with shorter side chains, such as α-amino-4-guanidino butyric acid ([Agb 7]NPFF) or phenylglycine ([Phg 8]NPFF), decreased efficacy for the NPFF₁ R to 25-31 % of the maximal response, suggesting that these agonist-receptor complexes are more susceptible to structural modifications. In contrast, mutations to the conserved Asp 6.59 residue in the third extracellular loop of both receptors revealed a higher sensitivity toward the hNPFF₂R receptor than toward hNPFF₁R. These data provide new insight into the subtype-specific agonistic activation of the NPFF₁ and NPFF(2) receptors that are necessary for the development of selective agonists.

Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

“…Purification and verification of the peptides were achieved as described previously (supplemental Table S1) (22).…”

“…Binding was determined with 1 nM N- [propionyl-3 H]hPrRP20 in the absence (total binding) or in the presence (non-specific binding) of 1 M unlabeled hPrRP20, respectively, as described previously (26,27). Our former evaluated protocol (28) Signal Transduction Assay-Signal transduction (inositol phosphate (IP) accumulation) assays were performed as described previously with minor modifications (22). The time of incubation was increased to 3 h for the double mutants of PrRPR and reduced to 1 h for measurement of concentrationresponse curves.…”

Pancreatic Polypeptide Is Recognized by Two Hydrophobic Domains of the Human Y4 Receptor Binding Pocket

“…Signal Transduction Assays-Signal transduction assays were performed on 24-or 48-well plates as described previously with minor changes (3,23). As transfection reagents, Metafectene and Metafectene Pro (Biontex) were used.…”

“…The NPYR is closely related to other class A GPCR families such as the neuropeptide FF receptor family and the orexin receptor family (3). NPY receptors are physiologically coupled to the G i or G o proteins; however, other reports show that rabbit Y 2 R and rabbit Y 4 R are also coupled to the G q protein, triggering an increase in inositol phosphate (4).…”

Does Kisspeptin Belong to the Proposed RF-Amide Peptide Family?

“…This turned out to be an ortholog of NPVF in humans (9, 47). Albeit with some cross-reactivity, NPFF has a high affinity to NPFFR2 (4) while NPVF more selectively activates NPFFR1 (6, 48, 49). …”