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

摘要

class="enumerated" style="list-style-type:decimal">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 [¹²⁵I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors.Specific binding of [¹²⁵I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with KD values of 0.026±0.006 and 0.57±0.14 nM respectively, and Bmax 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 KD 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 Gs or Gi.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.These studies indicate that [¹²⁵I]-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.