Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay

We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [ 35S]guanosine-5′- O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH 2 exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.