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RESEARCH ARTICLE
Murine GPRC6A Mediates Cellular Responses
to L-Amino Acids, but Not Osteocalcin
Variants
Patricia Rueda
1
, Elizabeth Harley
2
, Yao Lu
1
, Gregory D. Stewart
1
, Stewart Fabb
1
,
Natalie Diepenhorst
1
, Béatrice Cremers
2
, Marie-Hélène Rouillon
2
, Isabelle Wehrle
2
,
Anne Geant
2
, Gwladys Lamarche
2
, Katie Leach
1
, William N. Charman
1
,
Arthur Christopoulos
1
, Roger J. Summers
1
, Patrick M. Sexton
1
, Christopher
J. Langmead
1
*
1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville,
Victoria 3052, Australia, 2Institut de Recherches Servier, Suresnes, France
*chris.langmead@monash.edu
Abstract
Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein cou-
pled receptor is variously involved in regulation of metabolism, inflammation and endocrine
function. Such effects are described as mediated by extracellular calcium, L-amino acids,
the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introduc-
ing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated
by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A
are well established, verification of activity of osteocalcin at both human and mouse
GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro phar-
macology of mouse GPRC6A in response to its putative ligands in both recombinant and
endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide
(GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as ago-
nists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-
endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a
direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that
require GPRC6A may be indirect, rather than via direct activation of the receptor.
Introduction
GPRC6A is a class C G protein-coupled receptor (GPCR) that has been cloned from human,
mouse and rat. The receptor was initially deorphanised by fusion of its large N-terminal
domain to the heptahelical and C-terminal region of the related goldfish 5.24 receptor [1]. This
construct, and full length GPRC6A, mediates responses to L-amino acids, notably basic amino
acids such as arginine, ornithine and lysine. These ligands are thought to bind in the N-termi-
nal domain of the receptor [2] in a manner analogous to the binding of L-glutamate to
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 1/19
a11111
OPEN ACCESS
Citation: Rueda P, Harley E, Lu Y, Stewart GD, Fabb
S, Diepenhorst N, et al. (2016) Murine GPRC6A
Mediates Cellular Responses to L-Amino Acids, but
Not Osteocalcin Variants. PLoS ONE 11(1):
e0146846. doi:10.1371/journal.pone.0146846
Editor: Frederick G Hamel, Omaha Veterans Affairs
Medical Center, UNITED STATES
Received: August 27, 2015
Accepted: December 21, 2015
Published: January 19, 2016
Copyright: © 2016 Rueda et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Servier provided support in the form of
salaries for EH, BC, M-HR, IW, AG, GL and provided
research support to Monash University to fund
salaries for PR, YL, GDS, SF, ND and CJL. However,
Servier did not have any additional role in the study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. The specific
roles of these authors are articulated in the ‘author
contributions’section. AC and PMS are NHMRC
Principal Research Fellows.
metabotropic glutamate receptors. Small molecules, such as NPS-2143, calindol and indole-
based ligands, have been shown to bind in the 7TM domain of the receptor to act as negative
allosteric modulators of GPRC6A [3,4].
Recombinant GPRC6A expression studies have proven less than straightforward since the
human receptor expresses poorly at the cell surface, due to an intracellular retention motif in
the third intracellular loop [5]. Cell signalling studies with the murine orthologue suggest that
the receptor couples primarily via the Gα
q/11
pathway to increase inositol phosphate produc-
tion and mobilise intracellular calcium [1,6]; however, the efficiency of coupling is often poor
but can be substantially improved by co-expression of exogenous or mutated G proteins e.g.
Gα
qG66D
[7]. Other studies have indicated that GPRC6A may increase cyclic AMP and/or
phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) [8–12]. Thus the pre-
ferred downstream signal transduction pathway(s) of GPRC6A are not well defined and may
be dependent on species and cell background [7,13].
The pharmacology of GPRC6A is of interest due to recent studies that have implicated the
receptor in several metabolic, endocrine and inflammatory processes [11,12,14–19]. There
has been some debate as to the extent to which GPRC6A regulates metabolic function; one
Gprc6a KO mouse strain displays manifestations of metabolic syndrome, increased serum glu-
cose levels after an overnight fast as well as impaired insulin sensitivity [16]. However, a differ-
ent KO mouse displayed a subtler phenotype, with no evidence of impaired glucose handling
or insulin sensitivity (and disruptions in glucose metabolism only when the mice were fed a
high fat diet, a phenotype that could also result directly from obesity, rather than the absence
of GPRC6A itself) [18]. As a result, there remain questions as to the role of GPRC6A in con-
trolling metabolic and endocrine functions.
Additional interest in GPRC6A has emerged due to several studies reporting that it mediates
the metabolic and endocrine effects of the de-carboxylated form of the osteoblast-derived pep-
tide, osteocalcin (OCN). This peptide has been shown to control energy expenditure regulated
by the pancreas [20]; mice lacking OCN display decreased pancreatic cell proliferation, glucose
intolerance and insulin resistance. OCN, when injected into the peritoneal cavity of mice,
increases circulating insulin levels in WT but not in Gprc6a KO mice [12]. OCN has also been
shown to increase pancreatic β-cell proliferation, insulin release from pancreatic islets, insulin
sensitivity and energy expenditure and testosterone synthesis in the gonads in mice [20,21].
Furthermore, OCN-mediated insulin and glucagon-like peptide (GLP)-1 release have also been
reported in immortalised cell lines (e.g. pancreatic β-cells) and have been blocked by siRNA tar-
geting Gprc6a [12,22]. These data, together with the lack of OCN efficacy in Gprc6a KO mice,
both in vivo and ex vivo, has led to suggestions that de-carboxylated OCN acts directly via
GPRC6A to exert its metabolic and endocrine effects. Despite this evidence, a direct action of
OCN on the mouse receptor has been hard to confirm. Several studies have demonstrated that
de-carboxylated OCN can stimulate cAMP accumulation or ERK1/2 phosphorylation in
HEK293 cells recombinantly expressing GPRC6A (though the species tested is not always stated
[8,10–12]), suggesting that the signalling of the receptor may not be limited to Gα
q
linked path-
ways. However, another study in cells recombinantly expressing mouse GPRC6A failed to repli-
cate either the agonist activity of OCN, or the effects on cAMP signalling [13], leading the
authors to conclude that GPRC6A is primarily a Gα
q
-coupled, basic L-amino acid receptor.
The aim of this study was to determine whether de-carboxylated OCN is an agonist of
GPRC6A in either recombinant or endogenously expressing cells, using a range of cell signal-
ling and phenotypic assays. Lack of expression precluded study of the human receptor, but our
results in HEK293 cells suggest that murine GPRC6A is an L-amino acid receptor that prefer-
entially increases inositol phosphates and stimulates intracellular calcium mobilisation. No
cAMP signalling or ERK1/2 phosphorylation was evident, and no agonistic or modulatory
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 2/19
Competing Interests: EH, BC, M-HR, IW, AG, GL
are employees of Servier. This does not alter the
authors' adherence to PLOS ONE policies on sharing
data and materials.
effects of de-carboxylated OCN in any GPRC6A signalling assay were detected. We also con-
firmed an effect of L-ornithine to promote GLP-1 release and glucose-sensitive insulin secre-
tion in GPRC6A-expressing immortalised entero-endocrine and pancreatic β-cells,
respectively, but did not observe any effect of OCN variants. Only in rat insulinoma INS-1
(832) cells that did not express Gprc6a transcript were we able to observe any cellular response
to OCN variants, specifically a change in cellular impedance. Taken together, our results sug-
gest that the reported mouse GPRC6A-dependent effects of OCN may result from indirect,
rather than direct activation of the receptor.
Materials and Methods
Materials
RPMI 1640 (no glucose); Dulbecco’s Modified Eagle Medium (DMEM); DMEM (high glucose,
no glutamine, lysine, or arginine); sodium pyruvate; penicillin/streptomycin; foetal bovine
serum and lipofectamine LTX (GIBCO-Invitrogen-Life Technologies, Carlsbad, CA). β-Mer-
captoethanol, glucose, blasticidin S, ATP, forskolin, IBMX and L-amino acids (Sigma Aldrich,
St. Louis, MO); hygromycin B (Roche, Basel, Switzerland); tetracycline (Fluka, St. Gallen, Swit-
zerland); puromycin (InvivoGen (San Diego, CA); anti-cmyc 9E10 antibody (Santa Cruz, Dal-
las, Texas) and goat-anti mouse-PE antibody (GIBCO-Invitrogen-Life technologies, Carlsbad,
CA); SYTOX Red and Fluo-4 AM (Molecular Probes, Eugene, Oregon). GLUTag cells were
kindly provided by Dr. Daniel J. Drucker (Lunenfeld-Tanenbaum Research Institute, Mount
Sinai Hospital, Toronto, ON, Canada), MIN6 cells by Dr. Jun-ichi Miyazaki (Osaka University,
Osaka, Japan) and INS-1 (832) cells by Dr. Christopher Newgard (Sarah W. Stedman Nutrition
and Metabolism Center, Duke University, Durham, NC).
Osteocalcin (OCN) variants
Purified bovine OCN was purchased from Meridian Life Science (Memphis, TN, USA).
Human and mouse synthetic OCN (C-terminal acid and amide) were purchased from Federa-
tion Bioscience (Melbourne, VIC, Australia). Briefly, peptides were synthesised using Fmoc-
solid phase, purified, folded and the single disulphide bond formed by aeration. Although the
3D structure of the peptides obtained was not determined, the observed molecular weight was
consistent with the formation of the disulphide bond. Synthetic peptides were >98% pure as
assessed by reverse phase-HPLC and MALDI-TOF mass spectrometry. Mouse OCN was
expressed recombinantly as an N-terminal GST-tag fusion protein as previously described
[23]. Briefly, the fusion protein was expressed in BL21 (DE3) Codon Plus E.coli (Stratagene) in
2YT media after induction with 1 mM IPTG, 37°C for 3h. Cultures were harvested and pellets
were lysed using B-PER (Pierce). The soluble lysate fraction was collected after centrifugation
at 10000 g at 4°C for 15 min and purified using GST spin purification kits (Peirce). The fusion
protein was eluted using 3 mg/ml reduced glutathione, 125 mM Tris, 150 mM NaCl (pH 8.0)
and subsequently cleaved with thrombin (Novagen) according to the manufacturers protocol
overnight at 4°C with gentle rocking. The cleaved protein of interest was isolated using reverse
phase-HPLC (Lumina C8 (Phenoenex) column and a Prep LC system (Waters)). Mouse OCN
was eluted at 24 min (60% acetonitrile), confirmed by ESI-TOF mass spectrometry (Agilent
Technologies) and yields of 2mg/L culture were routinely achieved.
Cell line generation
Mouse and human GPRC6A (mGPRC6A and hGPRC6A) DNA sequences were obtained from
Genecopoeia in pDONR vectors. These clones, both with and without an N-terminal c-myc tag
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 3/19
preceded by the mGlu5 receptor signal peptide [13] (to detect receptor expression by immuno-
fluorescence), were assembled using PCR and transferred into the pCDNA 5/FRT/TO vector
(Invitrogen) or pEF-IRES-puro6 vector [24] (kindly provided by Sarawut Jitrapakdee, Depart-
ment of Biochemistry, University of Adelaide, Adelaide SA 5005). Gα
q
sequence in pcDNA3.1
+ was purchased from the cDNA Resource centre (Bloomsburg, Pennsylvania, USA) and sub-
stitution of Gly 66 for Asp was made using Quickchange II site-directed mutagenesis (Agilent
Technologies, USA) with the following primers; sense 5’-atgagaatcatccatgggtcag
attactctgatgaagataaaaggg-3’, antisense 5’- cccttttatcttcatcagagtaat
ctgacccatggatgattctcat-3’.
pcDNA 5/FRT/TO constructs were isogenically integrated into a FlpIn-TREx-HEK293 cell line
(Life Technologies). FlpIn-TREx-HEK293-mGPRC6A and FlpIn-TREx-HEK293-hGPRC6A cells
were then selected using 600 μg/ml hygromycin and 5 μg/ml blasticidin and grown in culture
medium consisting of Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% (v/v)
foetal bovine serum (FBS) at 37°C in 5% CO
2
and were sub-cultured in a 1:3 ratio every 3–4days.
Cells were cultured overnight in the presence of 1 μg/ml tetracycline to induce GPRC6A expression.
An additional, higher expressing mGPRC6A cell line was generated for GPRC6A assays in which
L-amino acids did not show activity (vide infra); pIRES-puro6 constructs were transfected into
FlpIn-TREx-HEK293 cells using Lipofectamine LTX following the manufacturer’s guidelines. 48 h
after transfection, cells were selected using 3 μg/ml puromycin and grown as above.
Flow cytometry
Cells were harvested with PBS-EDTA and 10
5
cells / well were seeded into a V-bottom 96-well
plate. Cells were incubated in blocking buffer (PBS with 0.01% azide, 5% goat serum, 2 mM
EDTA) for 30 min at 4°C. Primary antibody (anti-c-myc, 9E10) was added at a 1 μg/ml final
concentration and cells were incubated for 1 h at 4°C. After washing cells twice with wash
buffer (PBS, 0.01% sodium azide, 0.5% goat serum (v/v), 2 mM EDTA), cells were incubated
with 1 μg/ml of secondary antibody (goat anti-mouse-PE) diluted in wash buffer plus SYTOX
Red dead cell stain (1:1000; v/v) for 45 min at 4°C. Cells were then washed twice with wash
buffer and resuspended in PBS into BD Falcon tube with cell strainer cap. Cells were then ana-
lysed for PE and SYTOX Red fluorescence using a BD FACSCanto II flow cytometer. For anal-
ysis, the population of interest was gated on a FSC/SSC density plot. Live cells were selected
and analysed for PE stain intensity and/or percentage over untransfected control cells.
Quantitative PCR
Cells (cultured to 75% confluency and washed with PBS) or islets were treated with lysis buffer
from RNAeasy plus kit (Qiagen). RNA was extracted according to the manufacturer’s protocol.
Samples were eluted in 30 μl of ddH
2
O and RNA concentration determined using the Nano-
drop 2000 (Thermo Scientific). cDNA was synthesised from 1 μg total RNA using the M-MLV
RT polymerase (or water for the RT- controls) and random hexamers by incubating the sam-
ples at 60°C for 5 min followed by 1 h at 42°C and 15 minutes at 90°C. cDNA samples were
diluted 1 in 5 and 1 μl of each sample was used to perform real time PCR using Sybr Green or a
specific Taqman assay for mouse Gprc6a from Life technologies (Mm01192898_m1) using
mouse actin as a reference gene. Three known dilutions of plasmid containing the mouse or rat
Gprc6a sequence were run in parallel to determine the primer or Taqman assay efficiency. For
Sybr green assays, the primer pairs used were: mouseHprt: mHprt_f 5’ctggtgaaaaggac
ctctcg3’, mHprt_r 5’tgaagtactcattatagtcaagggca3’; mouseGprc6a:
mGprc6a_f 5’tcatgccacaggtgagttatgaat3’, mGprc6a_r 5’ggcaccaatccagt
tccatc3’; ratHprt: rHprt_f 5’ctggtgaaaaggacctctcg3’, rHprt_r 5’tgaagtgct
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 4/19
cattatagtcaagggca3’; ratGprc6a: rGprc6a_f 5’tcatgccacaggtgagttatgaat3’,
rGprc6a_r 5’ggcaccaacccagttccat3’. Results were expressed as ΔCt of the gene of
interest versus the reference gene for every sample.
Ca
2+i
mobilisation assays
FlpIn-TREx-HEK293-mGPRC6A cells were seeded into T75 flasks at 70% confluency. Cells
were transfected with 15μg of plasmid DNA (Gα
q
or Gα
qG66D
) with Lipofectamine LTX
according to manufacturer's instructions, then incubated for 24h at 37°C in 5% CO
2
. Cells
were plated into 96-well plates (10
5
cells/well) and equilibrated at 37°C in 5% CO
2
for 4-6h to
adhere, whereupon cells were serum starved overnight at 37°C in 5% CO
2
. For the assay, cells
were washed twice in Ca
2+
assay buffer (150 mM NaCl, 2.6 mM KCl, 1.2 mM MgCl
2
,10mM
dextrose, 10 mM HEPES, 2.2 mM CaCl
2
, 0.5% (w/v) BSA and 4 mM probenecid; pH 7.4).
Buffer was then replaced with Ca
2+
assay buffer containing 1μM Fluo-4-AM and incubated for
1h at 37°C in 5% CO
2
. Cells were washed twice more and replaced with Ca
2+
assay buffer at
37°C. Serial dilutions of test agonists were added by injection in a Flexstation™(Molecular
Devices) and fluorescence was measured at 485nm excitation and 520nm emission wave-
lengths. Agonist responses were normalised to the peak of response to 100 μM ATP.
Inositol phosphate accumulation assays
Inositol phosphate (IP
X
) accumulation experiments were performed using the IP-One HTRF
1
assay kit (Cisbio, France). Briefly, 24h after transfection with G proteins (as above), 10
5
cells
were seeded per well in PDL-coated, 96-well white plates and incubated overnight. Cells were
then washed 2 x 2h at 37°C in HBSS buffer (pH 7.2) containing 20 mM HEPES, 3.5 mM
NaHCO
3
, 1 mM CaCl
2
and 0.1% (w/v) BSA. Test compounds were then prepared in the kit
stimulation buffer containing 2 mM CaCl
2
; cells were then incubated with 70 μl of drugs for 30
min, after which 15 μl of both IP1-D2 and Ab-Cryp were added to the cells, followed by a 1 h
incubation at RT. Plates were read using an Envision plate reader (Perkin Elmer), using an
HTRF protocol. Results were calculated from the 665nm / 620nm HTRF ratio (Ratio = A
665nm
/
A
620nm
x10
4
) and expressed as a percentage of the maximal response obtained.
Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation assays
ERK1/2 phosphorylation time course experiments were performed in FlpIn-TREx-
HEK293-mGPRC6A cells transfected with G proteins (as above) using the SureFire Alphasc-
reen kit. Cells were plated in 96-well plates; after 6h incubation at 37°C in 5% CO
2
, cells were
washed twice with PBS and incubated in serum- and arginine / lysine-free DMEM at 37°C
overnight to reduce basal ERK1/2 phosphorylation. Cells were then stimulated with ligand at
various time points and incubated at 37°C in 5% CO
2
. 10% FBS (v/v) was used as a positive
control. The reaction was terminated by removal of compounds and lysis of cells with SureFire
lysis buffer (TGR Biosciences). The lysates were then treated as per the SureFire ERK1/2 kit
instructions. Plates were incubated in the dark at 37°C for 1 h, then left to equilibrate with the
ambient temperature before the fluorescence signal was measured using a Fusion plate reader
(PerkinElmer) with standard AlphaScreen settings. Data were normalised to the maximal
response elicited by 10% FBS (v/v) at the same time point.
Cyclic AMP accumulation assays
HEK293-mGPRC6A cells were seeded at a density of 10
5
cells per well into PDL-coated
96-well plates and incubated overnight in DMEM, supplemented with 10% FBS (v/v) at 37°C
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 5/19
in 5% CO
2
. Cells were then incubated in 90 μl of stimulation buffer (HBSS containing 5 mM
HEPES, 0.1% BSA (w/v), 0.5 mM IBMX; pH 7.4) for 30 min at 37°C. Test compounds (10 μlat
10 x final) were added and cells were incubated for an additional 30 min at 37°C. The medium
was removed and cells were incubated in 50 μl of ice-cold 100% ethanol. Once evaporated,
100 μl of lysis buffer (5 mM HEPES, 0.1% BSA (w/v), 0.3% Tween-20; pH 7.4) was added to
the cells and samples were stored at -20°C until further analysis. Samples were analysed using
the Alphascreen
1
cAMP assay kit (Perkin Elmer) following manufacturer instructions. Briefly,
10 μl of lysates were added to a 384-well Optiplate. After addition of 5 μl of acceptor bead mix,
plate was incubated at RT for 30 min followed by addition of 15 μl of donor bead mix. After
overnight (16 h) incubation at RT, plates were read using the Envision plate reader.
Label-free cell impedance assays
2.5 x 10
7
FlpIn-TREx-HEK293-mGPRC6A cells were seeded in a T175 flask and incubated
overnight at 37°C in 5% CO
2
. Cells were then transfected with 35 μg of plasmid DNA
(Gα
qG66D
) with Lipofectamine LTX according to manufacturer's instructions, then incubated
for 24h at 37°C in 5% CO
2
. Prior to the experiment, a background reading for each E-plate 96
(Bio-Rad, CA, USA) was measured after addition of 100 μl DMEM + 10% FBS per well. Cells
were recovered using Versene and seeded at a 10
5
cells/well density in 200 μl DMEM + 10%
FBS (containing 1 μg/ml tetracycline where indicated). Cells were washed 2 x 2 h in HBSS
buffer (pH 7.2) containing 20 mM HEPES, 1 mM CaCl
2
and 0.1% BSA (w/v) in a total volume
of 180 μl. Test compounds (20 μl at 10x final concentration) were added to the plate and cell
impedance was monitored for 3 h. Cell index was normalised to the buffer response (minus the
baseline response at zero time point).
For INS-1 cells, 100 μl of medium (RPMI 1640 no glucose, 10% FBS, 55 μMβ-mercap-
toethanol, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomy-
cin, 11.1 mM glucose) was added per well to an E-plate 96 and a background reading taken.
Cells were seeded at 2 x 10
4
/ well and the plates incubated in the instrument for 48 h. On the
day of the experiment, media was changed to EBSS buffer (180 μl; 2.8 mM glucose, 1.8 mM
CaCl
2
, 5.3 mM KCl, 0.8 mM MgSO
4
, 117 mM NaCl, 26 mM NaHCO
3
, 1 mM NaH
2
PO
4
). After
2 h incubation, cells were stimulated with 20 μl test compound (10 x final) prepared in the
same buffer. Data capture and analysis were as for HEK293 cells.
GLP-1 release assays
Intestinal L-cell model GLUTag cells were seeded in 24-well plates coated with Matrigel and
incubated for 6 days in DMEM with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin at
37°C in 5% CO
2
and grown for 4–5 days. Where indicated, cells were washed (1 or 2 x 1h) with
Krebs-HEPES buffer (20 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO
4
,5mM
NaHCO
3
, 1.2 mM KH
2
PO
4
, 2.5 mM CaCl
2
, 5.5 mM glucose, 0.2% BSA (w/v), pH 7.2) contain-
ing vildagliptin (300 μM) prior to addition of test compounds in the same buffer. After 2 h
incubation at 37°C in 5% CO
2
, the supernatant was recovered and centrifuged at 500 rpm for 5
min. Where used, NPS-2143 pre-treatment was for 30 min.
GLP-1 content in the cell supernatant was measured by ELISA (EGLP-35K, Millipore),
according to manufacturer’s instructions after overnight incubation of 15 μl supernatant in
100 μl Krebs-HEPES buffer at 4°C. After 5 x washes, 200 μl of detection conjugate was added
and incubated for 2 h at RT. After 3 x washes the reaction was developed with 200 μl of diluted
substrate (1:200) for 20 min and terminated by addition of 50 μl stop solution. After 5 min,
fluorescence was then measured using 355 nm excitation and 460 nm emission, transforming
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 6/19
the obtained signal into pmol GLP-1/well/ml and corrected for GLUTag cell protein content as
determined using the method of Bradford (1976).
Insulin release assays
MIN6 or βTC-6 cells were seeded in 24-well plates at 170,000 cells per well in DMEM (contain-
ing 4.5g/L glucose, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 15% de-
complemented FBS) and incubated at 37°C / 5% CO
2
for 72 h. Cells were then washed with
500 μl PBS and incubated in 500 μl deprivation media (DMEM with zero glucose, 10 mM
HEPES, 1 mM Na-pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 15% de-comple-
mented FBS) for 2 h at 37°C. Media was aspirated and after washing with 500 μl PBS, cells were
treated with 250 μl of Krebs buffer (250 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl
2
, 1.3 mM
CaCl
2
, 25 mM HEPES, 0.1% BSA (w/v), and 2 mM or 16.7 mM glucose; pH 7.4) in the presence
or absence of test compound. After incubation for 2 h at 37°C, the assay was terminated by
placing the plates on ice and supernatants were transferred to a 96-well plate in preparation for
insulin ELISA (Alpco) following manufacturer’s instructions. The cell monolayer was washed
twice with 500 μl of PBS and then lysed using 250 μl of 1 N NaOH prior to protein measure-
ment by the method of Bradford (1976).
Islets were collagenase-isolated from mice (10 x C57Bl/6; 12–13 weeks; Charles River) and
incubated overnight in RPMI (with 10% FBS (v/v), 10 mM HEPES, 100 U/ml penicillin,
100 μg/ml streptomycin, sodium pyruvate and 10 mM glucose) at 37°C in 5% CO
2
. Islets were
seeded into a 96-well plate (4 / well) in Kreb’s buffer containing 2.8 mM glucose and washed
twice with the same buffer. 200 μl test drugs were added in Kreb’s buffer with the required con-
centration of glucose and incubated at 37°C in 5% CO
2
for 90 min. The plate was centrifuged
for 10 s at 200 g, supernatant recovered and insulin levels detected as described above. This
protocol was approved by the IDRS (Institut de Recherches Servier) Institutional Animal Care
and Use Committee.
Data analysis
Concentration-response data were analysed using a three or four parameter logistic function
(Motulsky & Christopoulos, 2004) to generate estimates of agonist potency (LogEC
50
). Data
for all HEK293 assays represent the mean ± SEM of at least three independent experiments
(except cell impedance, n = 2). For GLP-1 release, the data represent mean ± SEM of three to
five independent experiments (except for OCN, wash; n = 2). For insulin release assays, data
represent the mean ± SEM of three independent experiments (except for islet studies; n = 2).
Insulin release data were normalised to the response to vehicle in either low or high [glucose].
Statistical analysis for GLP-1 release data were performed by one-way ANOVA, followed by
Sidak’s multiple comparisons test (treating wash / no wash datasets separately). Statistical anal-
yses for insulin release data were performed by two-way ANOVA (with [glucose] and drug
treatment as group variables), followed by Sidak’s multiple comparisons test. Where only high
[glucose] groups were evaluated, statistical analysis was by one-way ANOVA followed by
Sidak’s multiple comparisons test.
Results
Recombinant expression of GPRC6A orthologues
Using the tetracycline-inducible FlpIn technology, cells expressing both untagged and N-termi-
nally c-myc-tagged GPRC6A were generated in HEK293 cells. This resulted in a stable cell line
with robust cell-surface expression of mouse but not human GPRC6A, as determined by FACS
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 7/19
analysis (S1 Fig). Neither untransfected nor un-induced FlpIn-TREx-HEK293 cells showed
GPRC6A expression (S1 Fig and data not shown). Cell lines were also generated using a bicis-
tronic pIRES-puro6 expression vector expressing a puromycin resistance gene. Despite higher
expression for mouse GPRC6A compared to the FlpIn-TREx-HEK293 cell line, this approach
still failed to yield cell surface expression of the human receptor (S1 Fig). During the prepara-
tion of this manuscript, a study demonstrated, using chimeric human/mouse GPRC6A con-
structs, that a missing “RKLP”motif in the third intracellular loop causes the intracellular
retention and lack of cell-surface function of the human GPRC6A orthologue [5]. As a result of
the failure to express the human receptor at the cell surface, all subsequent functional studies
were performed in HEK293 cells stably expressing mouse GPRC6A.
Recombinant cell signalling
Since previous signalling studies suggest that GPRC6A preferentially couples to the Gα
q/11
pathway, we first assessed HEK293-mGPRC6A cells in assays of calcium mobilisation and ino-
sitol phosphate accumulation. In spite of robust receptor expression levels (S1 Fig), only very
small responses to L-amino acids were observed (data not shown). However, upon transient
expression of Gα
q
proteins (either WT or the promiscuous G66D mutant), robust agonist
activity to basic amino acids was detected in both calcium mobilisation and inositol phosphate
accumulation assays (Fig 1). Co-expression of Gα
qG66D
yielded a ten-fold higher potency
(pEC
50
= 4.0 ± 0.2) for L-ornithine in inositol phosphate accumulation assays compared to co-
expression of wild-type Gα
q
(pEC
50
= 3.0 ± 0.5; Fig 1A), likely reflecting improved stimulus-
response coupling. The observed signal was specific to GPRC6A, as G protein-transfected
parental HEK293 cells and non-tetracycline-induced FlpIn-TREx-HEK293-mGPRC6A cells
did not respond to L-amino acids (Fig 1B). Furthermore, inositol phosphate accumulation in
response to L-ornithine was sensitive to application of the GPRC6A antagonist, NPS-2143 (Fig
1C). In calcium mobilisation assays, the rank order of potency was L-ornithine >L-
arginine = L-lysine (Fig 1D), in agreement with previous studies [7], with little or no activity
detected for L-glutamine, L-valine or L-histidine.
Concomitantly to evaluating amino acid pharmacology of mGPRC6A, we profiled variants
of OCN in the assays. OCN contains three γ-carboxyglutamic acid residues, thus existing in
different forms based on the degree of carboxylation, which determines its affinity for the
extracellular bone matrix [25–27]. The degree of carboxylation could be a mechanism by
which the OCN function is controlled, although this theory is still not proven. Although in
mice the metabolic effects of OCN were observed exclusively when using the de-carboxylated
form, in middle-aged male subjects, levels of both de-carboxylated and carboxylated forms of
OCN are inversely associated with plasma glucose level, with the latter more closely related to
improved insulin sensitivity rather than increased β-cell function [28].
To obviate any species differences we profiled synthetic de-carboxylated human OCN, syn-
thetic and recombinant de-carboxylated mouse OCN (synthetic forms both acidic and ami-
dated) as well as partially carboxylated OCN from bovine bone (Table 1). Multiple OCN
variants were tested in the mGPRC6A inositol phosphate accumulation or calcium mobilisa-
tion assays (with both transiently transfected Gα
q
and Gα
qG66D
). As shown in Fig 2A (Gα
qG66D
transfected cells in inositol phosphate accumulation assays), no response was detected to any
OCN variant. Similar results were obtained in cells transiently transfected with Gα
q
and in cal-
cium mobilisation assays (data not shown). We then investigated the possibility of an indirect
effect of OCN on mGPRC6A function; however OCN variants (40 ng/ml) did not modulate
the potency or maximal response to L-ornithine in assays of inositol phosphate accumulation
(Fig 2B).
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 8/19
Fig 1. Calcium mobilisation and inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells. (A) Inositol phosphate accumulation
responses to L-ornithine in FlpIn-TREx-HEK293-mGPRC6A cells with co-expression of Gα
q
or Gα
qG66D
. (B) The L-ornithine response is specific to FlpIn-
TREx-HEK293 cells (co-expressing Gα
qG66D
) induced to express mGPRC6A with tetracycline, but absent in non-tetracycline induced or parental HEK293
cells. (C) GPRC6A-mediated inositol phosphate accumulation can be inhibited with the antagonist, NPS-2143 (30 μM). (D) Ca
2+
mobilisation induced by
various L-amino acids in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing Gα
qG66D
. The rank order of potency is L-ornithine >L-arginine = L-lysine
(values quoted represent pEC
50
±SEM). Similar responses were observed with Gα
q
co-expression (data not shown).
doi:10.1371/journal.pone.0146846.g001
Table 1. Osteocalcin (OCN) variants used in this study.
Species Method Source Carboxylation Status Sequence
Bovine Purified from
bone
Meridian Life
Science
Carboxylated / under-
carboxylated (Benton 1995,
Price 1976)
N/A
Human Recombinant Novus
Biological
De-carboxylated N/A [Cat. No. NP_954642.1; 1 a.a.—100 a.a.; includes ~ 26kDa N-terminal
GST tag]
Human Synthetic Federation
Bioscience
De-carboxylated (amide and
acid)
YLYQWLGAPVPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV-NH2/-
COOH
Mouse Recombinant Produced in
house
De-carboxylated GSPEF-YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI
doi:10.1371/journal.pone.0146846.t001
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 9/19
Although OCN variants did not display activity in assays classically linked to Gα
q
activation
in which L-amino acids displayed robust agonism, previous studies profiling OCN at GPRC6A
have also examined both cAMP accumulation and ERK1/2 phosphorylation. Therefore we
determined the effects of both L-ornithine and OCN variants in assays for these endpoints in
FlpIn-TREx-HEK293-mGPRC6A cells.
Despite robust increases in cAMP levels with forskolin in FlpIn-TREx-HEK293-mGPRC6A
cells, none of the putative GPRC6A agonists, including OCN variants, increased cAMP levels
in their own right. Furthermore, neither L-ornithine nor OCN variants inhibited cAMP levels
stimulated by forskolin (3 μM; Fig 3A). Similar results were observed in the higher-expressing
pIRES-puro6 cell line (S2 Fig). Furthermore, although the positive control (10% FBS) produced
a robust increase in ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells, none
of the putative GPRC6A agonists had any effect on ERK1/2 phosphorylation (in the absence or
presence of transiently expressed Gα
qG66D
;Fig 3B and 3C, respectively).
To date, GPRC6A signal transduction studies have been largely limited to one of the above
assay endpoints. To ensure that no hitherto unappreciated signalling pathway is activated
downstream of murine GPRC6A with OCN, we evaluated the ligand pharmacology in a label-
free assay of cellular impedance that represents a holistic measure of cellular activation. Using
real-time monitoring of impedance (xCELLigence), we profiled the pharmacology of
mGPRC6A expressed in FlpIn-TREx-HEK293 cells. Stimulation of endogenous P2Y puriner-
gic receptors with ATP was used as a control for detecting activation of Gα
q
protein-linked
pathway(s). When either untreated or tetracycline-induced FlpIn-TREx-HEK293-mGPRC6A
cells were stimulated with ATP, a concentration-dependent increase in cell index was observed,
with a similar maximal impedance and potency in both non-expressing and mGPRC6A-
expressing cells (Fig 4A). However, only mGPRC6A-expressing cells responded to L-ornithine
(pEC
50
= 4.2 ± 0.1; Fig 4B), with potency similar to that seen in inositol phosphate assays. Con-
sistent with observations in other assays, none of the OCN variants tested modulated cell
impedance in tetracycline-treated FlpIn-TREx-HEK293-mGPRC6A cells, suggesting that
OCN peptides do not activate murine GPRC6A (Fig 4C and data not shown).
Fig 2. Effect of OCN on inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells. (A) Variants of the putative GPRC6A peptide ligand
osteocalcin (OCN) fail to induce inositol phosphate accumulation in FlpIn-TREx-HEK293-mGPRC6A cells co-expressing Gα
qG66D
. (B) OCN variants (40 ng/
ml) do not modulate L-ornithine-stimulated inositol phosphate accumulation at mGPRC6A (OCN variantsas described in Table 1).
doi:10.1371/journal.pone.0146846.g002
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 10 / 19
GPRC6A function in entero-endocrine and pancreatic cells
Given the lack of response to OCN variants at recombinantly expressed mouse GPRC6A, we
postulated that this might be due to the lack of appropriate cellular machinery or adaptor pro-
teins in recombinant cells. Therefore we sought to test putative GPRC6A ligands in phenotypic
assays in immortalised murine cells and mouse pancreatic islets that express GPRC6A. The
GLUTag cell line was chosen as an entero-endocrine cell line as these cells have been previously
demonstrated to respond to L-amino acids to secrete glucagon-like peptide (GLP)-1. MIN6
and β-TC6 cells were chosen as prototypical immortalised pancreatic β-cell lines for the study
of insulin release. In parallel, we sought to evaluate insulin release in isolated mouse pancreatic
islets. The three cell lines (as well as the islets) were shown to express Gprc6a mRNA to varying
degrees (Fig 5). Finally we sought to identify a null cell line to demonstrate the specificity (or
lack thereof) to any peptide effects; using qPCR we could not detect any Gprc6a mRNA expres-
sion in INS-1(832) cells, a rat insulinoma cell line.
Fig 3. Modulation of cAMP levels and ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells. (A) OCN variants do not stimulate cAMP
accumulation in FlpIn-TREx-HEK293 cells stably expressing mGPRC6A (open circles). Neither L-ornithine nor OCN variants inhibit forksolin (3 μM)-
stimulated cAMP accumulation in the same cell line (filled circles; OCN variants as described in Table 1). (B) Neither L-ornithine (1 mM) nor OCN variants (40
ng/ml) stimulate ERK1/2 phosphorylation in FlpIn-TREx-HEK293-mGPRC6A cells; (C) a similar lack of activity was observed in cells co-expressing Gα
qG66D
.
doi:10.1371/journal.pone.0146846.g003
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 11 / 19
In GLUTag cells, L-ornithine (20 mM) significantly increased GLP-1 release, an effect that
was fully reversed by NPS-2143 (10 μM; Fig 6A). However, human synthetic OCN (acid form;
0.01–10 ng/ml) did not increase GLP-1 release whether the cells were washed (as for L-orni-
thine) or not (Fig 6A). A similar lack of effect was seen for the amidated form of the peptide
(data not shown). In MIN6 cells, glucose significantly promoted insulin secretion (P<0.0001,
two-way ANOVA) and L-ornithine (20 mM) significantly increased glucose-sensitive insulin
secretion (GSIS; P<0.01 vs. Vehicle, one-way ANOVA for high [glucose] followed by Sidak’s
multiple comparisons test; Fig 6B). This effect was fully reversed by NPS-2143 (10 μM; Fig 6B).
In contrast, mouse synthetic OCN (0.03–30 ng/mL) did not affect insulin secretion at either
glucose concentration (Fig 6B). A similar lack of effect was observed with recombinant mouse
OCN (0.03–30 ng/mL; data not shown). To ensure that the lack of effect was not cell line-
dependent, we also assessed OCN treatment on insulin release from β-TC6 cells and mouse
pancreatic islets. Although both preparations released insulin in a glucose-sensitive manner
(P<0.001, two-way ANOVA for [glucose] effect in both studies), OCN (0.03–100 ng/ml) had
no effect on insulin release at both glucose concentrations in either study (S3 Fig). The failure
of OCN variants to stimulate GPRC6A-linked phenotypic endpoints in GLUTag, β-TC6 or
MIN6 cells and mouse pancreatic islets casts further doubt as to whether GPRC6A is the direct
target of this peptide.
Finally we sought to examine the responsiveness of INS-1(832) cells, an immortalised rat
pancreatic β-cell line that does not express GPRC6A mRNA (as determined by qPCR; Fig 5).
For these studies we used the label-free xCELLigence assay to capture any cellular activity stim-
ulated by ligands. Interestingly, in this cell line we were able to detect significant changes in cell
impedance with purified bovine and synthetic mouse or human OCN (Fig 7). Thus, the only
functional assay in which we were able to detect a response to OCN variants was in a cell line
that does not appear to express GPRC6A.
Discussion
In these studies we present a broad range of experimental evidence suggesting that murine
GPRC6A is not a direct receptor target for the bone-derived peptide, osteocalcin (OCN). By
examining cell signalling events downstream of recombinantly expressed mouse GPRC6A and
Fig 4. Measurement of cellular impedance in FlpIn-TREx-HEK293-mGPRC6A cells. (A) ATP (acting via endogenous P2Y receptors) increases cellular
impedance in both tetracycline-induced and un-induced FlpIn-TREx-HEK293-mGPRC6A cells, whereas (B) L-ornithine increases cellular impedance only in
tetracycline-induced cells. (C) Bovine OCN did not change cell impedance in either induced or un-induced FlpIn-TREx-HEK293-mGPRC6A cells. A similar
lack of effect was shown for other OCN variants (data not shown).
doi:10.1371/journal.pone.0146846.g004
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 12 / 19
phenotypic assays in cell lines endogenously expressing the Gprc6a transcript, we confirmed
that GPRC6A is a receptor for basic L-amino acids, but were unable to detect any effect of
OCN variants, irrespective of peptide species or source. GPRC6A has emerged in recent years
as an interesting target due to its putative role in mediating the multiple in vivo effects of the
under- or de-carboxylated form of the bone-derived peptide, OCN. A number of studies have
demonstrated that OCN can increase pancreatic β-cell proliferation, insulin sensitivity and
energy expenditure in vivo, as well as promoting insulin secretion in vitro and ex vivo [12,20,
21]. Such effects may be beneficial in metabolic disorders such as type II diabetes, thus the
direct target for the actions of OCN may represent an attractive point of therapeutic interven-
tion. Furthermore, studies have shown that the beneficial effects of OCN are ameliorated or
absent in Gprc6a KO mice, thus implicating this class C GPCR as the direct target receptor for
OCN [12]. However, a definitive confirmation of this ligand-receptor pairing has proven some-
what elusive.
Whilst the ability of the receptor to be activated by basic L-amino acids is well accepted, sev-
eral studies have reported that OCN can activate GPRC6A to cause intracellular calcium mobi-
lisation [29], ERK1/2 phosphorylation [12], cAMP accumulation [8,30] and increased β-cell
proliferation [31] in GPRC6A-expressing cell lines or mouse pancreatic islets. The involvement
of GPRC6A has been shown by transfection of receptor-specific siRNA, receptor blockade with
a polyclonal antibody and, in the case of the islets, using tissue from KO mice.
A number of studies have specifically shown that OCN activates recombinantly expressed
GPRC6A; two studies show that OCN (at approx. 40–80 ng/ml) promote ERK1/2 phosphory-
lation in HEK293 cells expressing GPRC6A (species not stated; [10,12]). Interestingly, in one
study this response was sensitive to inhibition of phospholipase C and protein kinase C,
Fig 5. Determination of Gprc6a endogenous expression. Gprc6a mRNA expression in mouse primary
islets, murine β-TC6, GLUTag, MIN6 and rat INS-1(832) cells as determined by qPCR or Taqman; N.D. = not
detected.
doi:10.1371/journal.pone.0146846.g005
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 13 / 19
Fig 6. GPRC6A signalling in endogenously expressing cells. (A) L-ornithine (20 mM) significantly increases GLP-1 release from GLUTag cells
(**P<0.01 vs Vehicle, one-way ANOVA followed by Sidak’s multiple comparisons test). This effect is significantly reversed by NPS-2143 (^P<0.05 vs. L-
ornithine alone). Human synthetic OCN (0.01–10 ng/ml) did not significantly modulate GLP-1 release with or without cell washing. (B) High [glucose]
significantly enhanced insulin secretion by MIN6 cells (P<0.0001, two-way ANOVA). L-ornithine (20 mM), but not mouse synthetic OCN (acid form; 0.03–
100 ng/mL), significantly increased glucose-sensitive insulin secretion (**P<0.01 vs. Vehicle, one-way ANOVA for high [glucose] group followed by Sidak’s
multiple comparison test. ^^ P<0.01 vs. L-ornithine alone). Open bars, no glucose; filled bars, 16.7mM glucose. Data normalised to Insulin release in the
presence of 16.7mM glucose.
doi:10.1371/journal.pone.0146846.g006
Fig 7. Measurement of cellular impedance in Gprc6a-negative rat INS-1(832) cells. Concentration-dependent increases in cell index (a measure of
impedance) in INS-1(832) cells (that do not express Gprc6a) with synthetic human, purified bovine and synthetic mouse OCN. Statistical analysis performed
by two-way ANOVA ([OCN] and treatment); there was a significant effect of both [OCN] and treatment for all studies; *P<0.05, **P<0.01 vs. Vehicle by
Sidak’s multiple comparison test.
doi:10.1371/journal.pone.0146846.g007
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 14 / 19
suggesting that it may be downstream of Gα
q
activation. Other studies have showed that OCN
stimulates cAMP accumulation in HEK293 cells transfected with GPRC6A at a concentration
of either 3 ng/mL (species not stated for either receptor or peptide; [8]) or 60 ng/mL (mouse
GPRC6A with human OCN; [11])
More recently, Brauner-Osborne and colleagues sought to comprehensively delineate the
pharmacology of recombinantly expressed murine GPRC6A [13]. The major conclusions of
this study were discrepant with the observations described above insomuch as GPRC6A was
found to be primarily a Gα
q
coupled, basic L-amino acid receptor. No effects on cAMP levels
or ERK1/2 phosphorylation were observed and no agonist effect of OCN was evident in any of
the assays profiled [13]. Given these discrepancies, we sought to evaluate the pharmacology of
human and mouse GPRC6A in a range of cell signalling assays using L-amino acids and multi-
ple variants of OCN, including recombinant, purified and synthetic peptides. The aim of the
study was to provide a comprehensive evaluation of putative GPRC6A ligands in a range of
assays, including G protein-dependent signalling assays in cells recombinantly expressing
GPRC6A and metabolically-relevant phenotypic assays of GLP-1 release and insulin secretion
in immortalised entero-endocrine and pancreatic β-cells, respectively.
Consistent with recent data identifying an intracellular retention motif present in the third
intracellular loop of human GPRC6A [5], in our hands the human receptor expressed poorly at
the cell surface of FlpIn-TREx-HEK293 cells compared to mouse GPRC6A. Consequently, our
studies were limited to profiling this orthologue. Calcium mobilisation and inositol phosphate
accumulation studies confirmed that murine GPRC6A was activated by basic L-amino acids,
with L-ornithine, L-arginine and L-lysine the most potent agonists. However, robust inositol
phosphate and calcium responses were only observed with transient co-expression of Gα
q
or
the promiscuously coupling Gα
qG66D
. There was no detectable modulation of cAMP accumula-
tion or phosphorylation of ERK1/2 with L-ornithine, suggesting that mGPRC6A recombi-
nantly expressed in FlpIn-TREx-HEK293 cells is primarily a Gα
q
coupled receptor (Figs 3and
4). These data are largely concordant with the findings of Jacobsen et al. (2013) [13], who drew
a similar conclusion for the mouse GPRC6A recombinantly expressed in Chinese hamster
ovary cells (although they did detect ERK1/2 phosphorylation in response to L-amino acids).
The major finding of our recombinant studies was the lack of effect of OCN variants to
modulate any signalling pathway in FlpIn-TREx-HEK293 cells stably expressing mGPRC6A.
Under- or de-carboxylated forms of OCN purportedly mediate activity at GPRC6A. Therefore,
to obviate any issues of carboxylation status and/or species differences, we profiled multiple
OCN variants in our in vitro assays (as described in Table 1) over a concentration range previ-
ously reported to show activity. Nevertheless, no agonistic or modulatory activity was detected
at mGPRC6A, even in label-free assays of cell impedance that should capture any cellular sig-
nalling event (Fig 4B). Despite being performed in the same cell background (HEK293), these
data are clearly discrepant with the cAMP and ERK1/2 phosphorylation assays previously
reported for OCN at GPRC6A [8,12], though the species of the receptor tested was not rou-
tinely defined in the previous studies. This lack of OCN function concurs with the findings of
Jacobsen et al. (2013) [13], who detected only L-amino acid-mediated activation of
mGPRC6A.
Given the range of OCN variants profiled, we propose that species and/or carboxylation sta-
tus is unlikely to be a cause of the lack of activity in our assays. Other potential explanations
are that the species of GPRC6A is critical or that HEK293 cells lack the necessary scaffolding
and/or signalling proteins found in cells endogenously expressing GPRC6A. Unfortunately,
human GPRC6A was not expressed at the cell surface in our cells, precluding a study of its
pharmacology. However, we identified a range of immortalised murine entero-endocrine and
pancreatic β-cell lines that express Gprc6a mRNA. De-carboxylated, but not carboxylated
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 15 / 19
OCN has previously been shown to evoke GLP-1 release from the murine entero-endocrine
STC-1 cell line [22] and GPRC6A has been implicated in L-amino acid-mediated GLP-1
release from GLUTag cells, an immortalised murine intestinal L-cell line [32]. Our studies con-
firmed the presence of Gprc6a transcript and demonstrated L-ornithine stimulated GLP-1
release from GLUTag cells in a NPS-2143-sensitive manner, indicative of a GPRC6A-mediated
mechanism (Fig 6A). However, OCN treatment failed to increase GLP-1 release from this cell
line. OCN has also been shown to increase insulin secretion from pancreatic islets [31] and
engender ERK1/2 phosphorylation in βTC-6 cells [12], both in a GPRC6A-dependent manner.
We confirmed the expression of Gprc6a transcript in the pancreatic β-cell lines MIN6 and
βTC-6, as well as in mouse pancreatic islets (Fig 5). MIN6 cells displayed glucose-sensitive
insulin secretion (GSIS), which could be increased by L-ornithine in an NPS-2143-senstive
manner (Fig 6B). However, OCN treatment failed to induce GSIS, a profile that was replicated
in both βTC-6 cells and isolated mouse pancreatic islets (S1 Fig). Thus we have been unable to
replicate any of the reported GPRC6A-dependent in vitro activities of OCN.
Interestingly, the only cell line in which we could detect a cellular response to OCN variants
was the rat insulinoma INS-1(832) cell line, in which bovine, mouse and human OCN dis-
played significant, concentration-dependent activity in the label-free cellular impedance assay
as applied to the FlpIn-TREx-HEK293-mGPRC6A cells (Fig 7). However, no Gprc6a mRNA
was detected in INS-1(832) cells by quantitative PCR. Whilst these data cannot prove that
OCN is not a direct agonist of GPRC6A, it remains interesting that the only cell line in which
our studies could detect effects of OCN is one in which there is no detectable transcript for the
receptor.
In summary our studies have not been able to verify the purported agonist activity of OCN
at mouse GPRC6A; in vitro cell signalling and phenotypic assays support the notion that
murine GPRC6A is primarily a receptor for basic L-amino acids that signals via Gα
q
proteins,
in general agreement with the data of Jacobsen et al. (2013) [13]. The most parsimonious expla-
nation for the data is that OCN does not directly activate mouse GPRC6A and that the
observed in vivo effects of OCN reported in the literature may depend on GPRC6A, but are
indirect and not due to an interaction with the receptor. This would account for many of the
OCN effects being absent in Gprc6a KO mice. For the small number of reports of direct phar-
macological effects of OCN on recombinantly expressed GPRC6A in HEK293 cells, as dis-
cussed above, only in one study is the species stated. It is possible that the human or rat
receptor respond differently to OCN in vitro compared to the mouse clone that has been the
focus of this and other studies [13]. Overall, our data do not support the notion of OCN as a
direct agonist of murine GPRC6A, suggesting that the receptor may not represent a suitable
therapeutic target to replicate the metabolically favourable effects of OCN in vivo.
Supporting Information
S1 Fig. Cell surface expression of mouse and human GPRC6A. FACS expression analysis
with anti-c-myc (9E10) staining of FlpIn-TREx-HEK293 stably transfected with human or
mouse c-myc GPRC6A using the pcDNA 5/FRT/TO or pIRES-puro6 expression constructs.
Expression assessed as % of GPRC6A-expressing cells and total GPRC6A expression (fluores-
cence). Human GPRC6A did not express at the cell surface; the mouse orthologue was moder-
ately expressed using the pcDNA 5/FRT/TO construct and at higher levels using the pIRES-
puro6 vector.
(TIF)
S2 Fig. Measurement of cAMP modulation in HEK293-pIRES-puro6-mGPRC6A cells. Nei-
ther L-ornithine nor OCN variants stimulate cAMP accumulation in HEK293 cells stably
Murine GPRC6A Responds to L-Amino Acids, but Not Osteocalcin
PLOS ONE | DOI:10.1371/journal.pone.0146846 January 19, 2016 16 / 19
expressing pIRES-puro6-mGPRC6A (open circles); none of the ligands inhibit forksolin
(3 μM)-stimulated cAMP accumulation in the same cell line (filled circles). OCN variants are
as described in Table 1
(TIF)
S3 Fig. Insulin secretion by β-TC6 cells or mouse pancreatic islets. (A) Glucose significantly
enhanced insulin secretion by β-TC6 cells (P<0.0001, two-way ANOVA), but there was no
significant effect of either L-ornithine (20 mM) or human synthetic OCN (acid form; 0.03–100
ng/ml) to increase GSIS. (B) High [glucose] significantly enhanced insulin secretion by mouse
pancreatic islets (P<0.0001, two-way ANOVA). L-arginine (20 mM), but not human syn-
thetic OCN (0.03–100 ng/ml) significantly increased GSIS (P<0.05 vs. Vehicle, two-way
ANOVA followed by Sidak’s multiple comparisons test). Open bars, no glucose; filled bars,
16.7mM glucose.
(TIF)
S1 Table. Summary of functional assays performed in cell lines recombinantly or endoge-
nously expressing GPRC6A.
(DOCX)
Author Contributions
Conceived and designed the experiments: PR EH GDS CJL PMS AC RJS. Performed the exper-
iments: PR YL GDS SF ND BC M-HR IW AG GL KL. Analyzed the data: PR YL GDS SF EH
CJL. Wrote the paper: PR CJL EH WNC AC RJS PMS.
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