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

Selectivity is a major issue in closely related multiligand/multireceptor systems. In this study we investigated the RFamide systems of hNPFF 1 R and hNPFF 2 R 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 NPFF 1 R and NPFF 2 R. However, the C‐terminal Phe 8 was able to be replaced by Trp or His with only a minor loss in potency at the NPFF 2 R relative to the NPFF 1 R. 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 NPFF 1 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 hNPFF 2 R receptor than toward hNPFF 1 R. These data provide new insight into the subtype‐specific agonistic activation of the NPFF 1 and NPFF 2 receptors that are necessary for the development of selective agonists.