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. 2013 Nov;347(2):298-309.
doi: 10.1124/jpet.113.206276. Epub 2013 Sep 5.

Delineation of the GPRC6A receptor signaling pathways using a mammalian cell line stably expressing the receptor

Stine Engesgaard Jacobsen  1 , Lenea Nørskov-Lauritsen, Alex Rojas Bie Thomsen, Sanela Smajilovic, Petrine Wellendorph, Niklas H P Larsson, Anders Lehmann, Vikram Kjøller Bhatia, Hans Bräuner-Osborne
Affiliations

Delineation of the GPRC6A receptor signaling pathways using a mammalian cell line stably expressing the receptor

Stine Engesgaard Jacobsen et al. J Pharmacol Exp Ther. 2013 Nov.

Abstract

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-α-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 Gq, Gs, Gi, and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic l-α-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-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 Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in high-throughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.

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Figures

Fig. 1.
Fig. 1.
Structures of previously reported GPRC6A ligands. (A) The amino acid l-ornithine; (B) the steroid testosterone; (C) the peptid osteocalcin (porcine) (adapted from Protein Data Bank code 1Q8H); (D) the GPRC6A-selective antagonist compound 1; and (E) the amino acid sequence of bovine osteocalcin (bOC) and the differences compared with porcine osteocalcin (pOC).
Fig. 2.
Fig. 2.
Evaluation of cell lines stably and transiently expressing GPRC6A. (A) RT-PCR performed on total RNA extracted from the mGPRC6A-CHO and Flp-In-CHO cell lines. Water was used as a negative control. Bands were detected by using intron-spanning primers specific for (top) mGPRC6A and (bottom) the ubiquitously, endogenously expressed GAPDH. Only the shown bands were detected. (B) ELISA to detect cell-surface expression of c-myc tagged GPRC6A in the mGPRC6A-CHO, hGPRC6A-CHO, and Flp-In-CHO cell lines, and in tsA201 cells transiently expressing mGPRC6A alone, mGPRC6A and Gq(G66D), or empty vector alone. Surface-expressed receptors were detected using c-myc mouse monoclonal antibody and goat anti-mouse IgG HRP conjugate. Luminescence was measured after addition of a chemiluminescent HRP substrate. Data are shown as means ± S.E.M. of at least three independent experiments performed in triplicate. (C) Measurements of intracellular Ca2+ release. Responses to l-Orn and ATP were measured in mGPRC6A-CHO using the Fluo-4 NW calcium assay kit. Data are ΔRFU (peak fluorescence units after agonist addition subtracted fluorescence before agonist addition) and are shown as means ± S.E.M. of two independent experiments performed in triplicate. (D) Measurements of IP1 accumulation in response to 1 mM l-Orn as assessed by the HTRF IP-One assay. Responses were measured in the mGPRC6A-CHO, hGPRC6A-CHO, and Flp-In-CHO cell lines and in tsA201 cells transiently expressing mGPRC6A alone, mGPRC6A and Gq(G66D), or empty vector alone. Data are means ± S.E.M. of at least three independent experiments performed in triplicate.
Fig. 3.
Fig. 3.
Activity testing in the Gq signaling pathway. (A) Measurements of IP1 accumulation as a result of Gq activation. Responses to the ligands l-Orn, Ca2+, testosterone, and osteocalcin were measured in mGPRC6A-CHO and Flp-In-CHO cells, respectively, by means of the HTRF IP-One assay. Data are means ± S.E.M. of four independent experiments performed in triplicate. Significant differences from basal (ligand buffer) were calculated by performing a one-way ANOVA followed by Dunnett’s post-test (*P < 0.05, ***P < 0.001). (B) Activity testing of three different commercially available recombinant forms of osteocalcin using the IP turnover assay. Osteocalcin were tested in the presence of EC25 of l-Orn in tsA201 cells cotransfected with mGPRC6A and Gq(G66D). Results are shown as CPM and are means ± S.D. of a single representative experiment performed in duplicate. An additional experiment gave similar results. Significant differences from basal (ligand buffer) were calculated by performing a one-way ANOVA followed by Dunnett’s post-test (not significant, P > 0.05).
Fig. 4.
Fig. 4.
Validation of mGPRC6A pharmacology as mediated by the Gq signaling pathway. (A, B, and C), concentration-response curve of l-Orn using mGPRC6A-CHO cells in the presence and absence of (A) the GPRC6A-selective antagonist compound 1, (B) the specific Gq inhibitor UBO-QIC, or (C) the specific PI-PLC inhibitor U73122. Responses are shown as IP1 accumulation measured by the HTRF IP-One assay. Data are means ± S.D. of a single representative experiment performed in triplicate. Two additional experiments gave similar results. (D) Ca2+-induced IP1 production in mGPRC6A-CHO and Flp-In-CHO cells in the presence and absence of compound 1. Data are normalized to the basal level of IP1 in ligand buffer and are shown as means ± S.E.M. of three independent experiments performed in triplicate. Statistical comparison was performed within each group (each cell line) by using an unpaired student’s t test (* P < 0.05). (E) The basal level of IP1 in ligand buffer in the Flp-In-CHO cell line in the presence and absence of 1 µM UBO-QIC. Data are means ± S.E.M. of three independent experiments performed in triplicate. Statistical comparison was performed by using an unpaired student’s t test (**P < 0.01).
Fig. 5.
Fig. 5.
Measurements of (A) cAMP accumulation as a result of Gs activation and (B) cAMP inhibition as a result of Gi activation. Responses to ATP, forskolin, l-Orn, Ca2+, testosterone, and osteocalcin were measured in mGPRC6A-CHO and Flp-In-CHO cells, respectively, by using the HTRF cAMP assay. (A) Data are means ± S.E.M. of three independent experiments performed in triplicate. Significant differences from basal (ligand buffer) were determined by performing a one-way ANOVA followed by Dunnett’s post-test (***P < 0.001). (B) Data are normalized to the cAMP production in response to 20 µM forskolin and are shown as means ± S.E.M. of five independent experiments performed in triplicate. Significant differences from basal (20 µM forskolin) were determined by performing a one-way ANOVA followed by Dunnett’s post-test (**P < 0.01, ***P < 0.001).
Fig. 6.
Fig. 6.
Measurements of ERK activation in mGPRC6A-CHO and Flp-In-CHO cells on stimulation with (A) 10 mM l-Orn, (B) 1 mM l-Orn, (C) 10 mM Ca2+, (D) 80 nM testosterone, and (E) 0.01 µM osteocalcin. ERK activation was assessed by using antibodies against phosphorylated ERK (P-ERK) and total ERK (T-ERK) after Western blotting. Quantification of the western blots has been performed by using the ImageJ program (http://rsbweb.nih.gov/ij/download.html) and by normalizing the P-ERK response to the corresponding T-ERK response. Furthermore, the data have been normalized to the basal level of P-ERK/T-ERK in ligand buffer and are shown as means ± S.E.M. of (A, B, C) three or (D, E) two independent experiments. Statistical comparison between basal (ligand buffer) and the ligand was performed within each group (each cell line) by using an unpaired student’s t test (**P < 0.01, ***P < 0.001).
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