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

Br J Pharmacol. 2000 Oct;131(4):683-8. doi: 10.1038/sj.bjp.0703617.

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding, Competitive
  • Calcium / metabolism
  • Cells, Cultured
  • Cyclic AMP / biosynthesis
  • Humans
  • Hypothalamic Hormones / metabolism*
  • Hypothalamic Hormones / pharmacology
  • Iodine Radioisotopes
  • Kinetics
  • Molecular Sequence Data
  • Neuropeptides / metabolism*
  • Neuropeptides / pharmacology
  • Prolactin-Releasing Hormone
  • Rats
  • Receptors, Cell Surface / metabolism*
  • Receptors, G-Protein-Coupled*

Substances

  • Hypothalamic Hormones
  • Iodine Radioisotopes
  • Neuropeptides
  • PRLH protein, human
  • PRLHR protein, human
  • Prlh protein, rat
  • Prolactin-Releasing Hormone
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Cyclic AMP
  • Calcium