Monitoring Gq-coupled receptor response through inositol phosphate quantification with the IP-One assay

The GPRC6A receptor is a recently "deorphanized" class C G protein-coupled receptor. We and others have shown that this receptor is coactivated by basic L-a-amino acids and divalent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the G q , G s , G i , and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic L-a-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the G q pathway. We were not able to confirm previously published data demonstrating G i -and G s -mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of G q activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in highthroughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.

Section: Delineation Of the Gprc6a Receptor Signaling Pathways Resultsmentioning

confidence: 99%

Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

Jacobsen

Nørskov-Lauritsen

Thomsen

et al. 2013

“…hPTH1R-mediated stimulation of inositol monophosphate (IP 1 ) accumulation was measured using the CisBio IPOne TRF (time-resolved fluorescence) assay kit (Bedford, MA) (Trinquet et al, 2011) and using the same CHO-K1 clone stably expressing recombinant hPTH1R used for cAMP accumulation assays (DiscoveRX). In brief, 100-ml aliquots of cells (30,000 cells/well) were added to 96-well assay microtiter plates and incubated for 48 hours at 37°C (5% CO 2 , 95% RH).…”

Section: Methodsmentioning

confidence: 99%

Parathyroid Hormone (PTH) and PTH-Related Peptide Domains Contributing to Activation of Different PTH Receptor–Mediated Signaling Pathways

Cupp¹,

Nayak²,

Adem³

et al. 2013

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), acting through the osteoblast PTH1 receptor (PTH1R), play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation, whereas continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including G s /cAMP/protein kinase A, G q /calcium/protein kinase C, b-arrestin recruitment, and extracellular signal-related kinase (ERK)1/2 phosphorylation, as well as receptor internalization, but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal human PTH1R (hPTH1R) functional assays capturing G s -and G q -signaling, b-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization to further define the patterns of PTH1R signaling that they stimulate and further establish peptide domains contributing to agonist activity. Results indicate that both Nand C-terminal domains of PTH and PTHrP are critical for activation of signaling pathways. However, modifications of both regions lead to more substantial decreases in agonist potency and efficacy to stimulate G q -signaling, b-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than to stimulate G s -signaling. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptides evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.

“…We used a homogeneous time-resolved fluorescence-based (HTRF) assay kit to detect inositol monophosphates derived from the breakdown of inositol 1,4,5, trisphosphate (Trinquet et al, 2011). HEK-293T cells were transiently cotransfected with human, mouse, or rat orthologs of FLAG-GPR35-eYFP and a chimeric G protein (Ga q -13 ) (Jenkins et al, 2012) using polyethylenimine.…”

Section: Methodsmentioning

confidence: 99%

High-Throughput Identification and Characterization of Novel, Species-selective GPR35 Agonists

Neetoo-Isseljee

Mackenzie

Southern

et al. 2012

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter b-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both b-arrestin recruitment and G proteindependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between b-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease.