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 peptide (PrRP) has been identi®ed as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [ ]-PrRP-20 was saturable, and analysis suggested evidence of both high and low anity 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 8570+2240 fmol mg protein 71 respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K D of 0.012 nM. 3 Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high anity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any anity for GPR10. 4 Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular 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 con®rmed 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 Ca 2+ stores. 6 These studies indicate that [ 125 I]-PrRP-20 is a speci®c, high anity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

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