Functional differences between NPFF1 and NPFF2 receptor coupling:: High intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPγS binding

By using an optimized [S-35]GTP gamma S binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF1 and NPFF2 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF1 and NPFF2 receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF2 receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF1 receptors, NPFF2 receptors exhibited an extraordinary activity since all compounds tested displayed EC50 values of GTP gamma S binding lower than their K, values. Comparisons of functional values between NPFF1 and NPFF, receptors revealed unexpected potent selective NPFF2 agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that GOCD and Gas are the main transducers of NPFF1 receptors while NPFF2 are probably coupled with G alpha(i2), G alpha(i3), G alpha(o) and G alpha(s) proteins. Our data indicate that NPPF1 and NPFF2 receptors are differently coupled to G proteins in CHO cells. (c) 2006 Elsevier Ltd. All rights reserved.