title: Characterization of the binding of [(125)I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor.
abstract: 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 identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-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 8570+/-2240 fmol mg protein(-1) 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. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity 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 affinity for GPR10. 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). 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 Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity 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.
authors.affiliation: Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, Essex, CM19 5AW. Christopher_J_Langmead@sbphrd.com
authors.forename: C J
authors.initials: CJ
authors.lastname: Langmead
authors.forename: P G
authors.initials: PG
authors.lastname: Szekeres
authors.forename: J K
authors.initials: JK
authors.lastname: Chambers
authors.forename: S J
authors.initials: SJ
authors.lastname: Ratcliffe
authors.forename: D N
authors.initials: DN
authors.lastname: Jones
authors.forename: W D
authors.initials: WD
authors.lastname: Hirst
authors.forename: G W
authors.initials: GW
authors.lastname: Price
authors.forename: H J
authors.initials: HJ
authors.lastname: Herdon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D007028:Hypothalamic Hormones / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism* / Q000494:pharmacology; D056690:Prolactin-Releasing Hormone; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled
chemical_list: D007028:Hypothalamic Hormones; D007457:Iodine Radioisotopes; D009479:Neuropeptides; C534377:PRLH protein, human; C112345:PRLHR protein, human; C534379:Prlh protein, rat; D056690:Prolactin-Releasing Hormone; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D002118:Calcium