Characterization of the binding of [I-125]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor

1 GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing p eptide (PrRP) has been identified as an endogenous ligand for GPR10, and oc curs as 31 and 20 amino acid forms. The present study characterizes the bin ding of [I-125]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. 2 Specific binding of [I-125]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K-D values of 0.026+/-0 .006 and 0.57+/-0.14 nM respectively, and B-max values of 3010+/-400 and 85 70+/-2240 fmol mg protein-L respectively. Kinetic studies were unable to di stinguish two sites, but single site analysis of association and dissociati on data produced a K-D of 0.012 nM. 3 Competition studies revealed that human and rat PrRP-20 and PrRP-31 all d isplay high affinity for GPR10. A range of other drugs which are known liga nds at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstra ted any affinity for GPR10. 4 Human PrRP-20 failed to alter basal or forskolin-stimulated levels of int racellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). 5 Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca2+ stores. 6 These studies indicate that [I-125]-PrRP-20 is a specific, high affinity radioligand for GPR1. The availability of this radioligand binding assay wi ll be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.