Functional differences between NPFF 1 and NPFF 2 receptor coupling: High intrinsic activities of RFamide-related peptides on stimulation of [ 35S]GTPγS binding

Abstract

By using an optimized [ 35S]GTPγS 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 NPFF 1 and NPFF 2 receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF 1 and NPFF 2 receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF 2 receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF 1 receptors, NPFF 2 receptors exhibited an extraordinary activity since all compounds tested displayed EC 50 values of GTPγS binding lower than their K I values. Comparisons of functional values between NPFF 1 and NPFF 2 receptors revealed unexpected potent selective NPFF 2 agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Gα i3 and Gα s are the main transducers of NPFF 1 receptors while NPFF 2 are probably coupled with Gα i2, Gα i3, Gα o and Gα s proteins. Our data indicate that NPPF 1 and NPFF 2 receptors are differently coupled to G proteins in CHO cells.