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

Abstract

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.

The prolactin-releasing peptide antagonizes the opioid system through its receptor GPR10

“…The situation is somewhat more complex for CCK receptors, which have been shown to couple both to G q and G i family members. GPR10 has also been reported to couple to the G q class of G proteins in some cell types 35 . The interference of anti-opioid systems with opiate peptide signaling therefore probably involves their action at different sites of the neuronal network, although interference between two receptors expressed in the same cell has been proposed as well 6 .…”

“…Coronal cryostat brain sections (15 mm) were hybridized with 3¢-end-labeled [a 35 S]dATP oligonucleotide probes for proenkephalin and pronociceptin as described in the Supplementary Methods. Quantification was performed for areas showing moderate to high expression levels.…”

Prolactin Releasing Peptide Has High Affinity and Efficacy at Neuropeptide FF2 Receptors

“…Taken together, both the hNPFF2 and the hPrRP receptor seem to couple to the G i/o class of G proteins in CHO cells. It has previously been suggested that the hPrRP receptor /GRP10 signals through the G q pathway in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary, but the coupling clearly depends on the specific cellular system in which the receptor is expressed (Kimura et al, 2000;Langmead et al, 2000).…”

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

“…S2). Functional coupling of GPR10 to Gprotein subunit Gq and to intracellular calcium is known to occur (28). Familial mutations to the GPR10 gene resulting in altered calcium homeostasis have also been reported (29).…”