Molecular and Cellular Endocrinology 348 (2012) 247–254

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

The truncated ghrelin receptor polypeptide (GHS-R1b) is localized

in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors
(GHS-R1a) to attenuate their cell surface expression
Kevin B.S. Chow, Jingxin Sun, Kit Man Chu, Wing Tai Cheung, Christopher H.K. Cheng, Helen Wise ⇑
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

a r t i c l e i n f o a b s t r a c t

Article history: The ghrelin receptor (GHS-R1a) is remarkable amongst G-protein-coupled receptors for its high degree of
Received 22 July 2011 constitutive activity, and this agonist-independent activity may be important for its physiological func-
Received in revised form 24 August 2011 tion in the control of food intake and body weight. Ghrelin receptors form heterodimers with the trun-
Accepted 26 August 2011
cated ghrelin receptor polypeptide (GHS-R1b), which has a dominant-negative effect on ghrelin
Available online 31 August 2011
receptor function. Here we show that GHS-R1b has an intracellular localization distinct from ghrelin
receptors, being primarily localized in the endoplasmic reticulum. Immunocytochemical studies suggest
Keywords:
that GHS-R1b decreases the plasma membrane expression of ghrelin receptors, but the overall distribu-
Ghrelin receptor
GHS-R1b
tion profile of ghrelin receptors in isolated subcellular fractions is unaffected by GHS-R1b. Using biolumi-
Dominant-negative mutant nescence resonance energy transfer methods, we have shown that while ghrelin receptor homodimers
Heterodimerization are evenly distributed in all subcellular fractions, GHS-R1a/GHS-R1b heterodimers are concentrated
within the endoplasmic reticulum and these results suggest that GHS-R1b traps ghrelin receptors within
the endoplasmic reticulum by the process of oligomerization. Furthermore, ghrelin receptors constitu-
tively activated extracellular signal-regulated kinases 1/2 in the endoplasmic reticulum, but this small
response was not affected by GHS-R1b and its physiological relevance is uncertain. Taken together, these
results suggest that ghrelin receptors can be retained in the endoplasmic reticulum by heterodimeriza-
tion with GHS-R1b, and constitutive activation of phospholipase C is attenuated due to decreased cell sur-
face expression of ghrelin receptors. However, sufficient ghrelin receptor homodimers can still be
expressed on the cell surface for maximal responses to agonist stimulation.
Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Ghrelin is a gastrointestinal hormone and is the only circulating
orexigenic hormone secreted from a peripheral organ to act on the
hypothalamic arcuate and ventromedial nuclei to regulate appetite
(Jia et al., 2008; Kojima and Kangawa, 2010). The ghrelin receptor
(growth hormone secretagogue receptor type 1a: GHS-R1a) is a G-
protein-coupled receptor (GPCR) with a high degree of constitutive
activity in vitro (Holst et al., 2003; Leung et al., 2007) and this ago-
nist-independent activity may confer some anti-apoptotic proper-
ties to cells (Lau et al., 2009). Two novel variants of the GHSR gene
have been identified which produced a selective loss of ghrelin
receptor constitutive signalling and defects in growth profiles
(Pantel et al., 2006). High constitutive signalling from ghrelin
receptors may be important for their in vivo function in the control
of food intake and body weight, as recently demonstrated using a
potent nverse agonist for the ghrelin receptor (Petersen et al.,
⇑ Corresponding author. Tel.: +852 2609 6849; fax: +852 2603 5139.
E-mail address: helenwise@cuhk.edu.hk (H. Wise).
2009). Nevertheless, studies of mice lacking ghrelin and the ghre-
lin receptor indicate that the primary metabolic function of ghrelin
in adult mice may be to modulate glucose sensing and insulin sen-
sitivity, rather than directly regulate energy intake and energy
expenditure (Sun et al., 2008). Indeed, the role of ghrelin in insulin
regulation has been strengthened by observations that mice defi-
cient in acylated ghrelin experience life-threatening hypoglycae-
mia which can be prevented by exogenous ghrelin or growth
hormone (Zhao et al., 2010).
The constitutive activity of ghrelin receptors appears to be nec-
essary for effective ghrelin receptor internalization to recycling
compartments (Holliday et al., 2007), and in HEK293 cells tran-
siently expressing ghrelin receptors, the constitutive activity of
ghrelin receptors is dramatically affected by co-expression with
the truncated ghrelin receptor polypeptide (GHS-R1b) due to de-
creased cell surface expression (Chan and Cheng, 2004; Chu
et al., 2007; Leung et al., 2007). GHS-R1b is a naturally occurring
truncated splice variant consisting of the first five predicted trans-
membrane regions of the ghrelin receptor; it fails to bind ghrelin
and has no known signalling activity (Howard et al., 1996). Relative

0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2011.08.034
248 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254
expression levels of ghrelin receptor and GHS-R1b mRNA vary in
different tissues (Gnanapavan et al., 2002), and in somatotroph
adenomas there are significantly higher levels of GHS-R1b mRNA
compared with normal pituitary tissues (Korbonits et al., 2001),
suggesting that GHS-R1b may be oncogenic in these tissues. In hu-
man carotid artery endothelial cells GHS-R1b mRNA expression is
significantly greater than in smooth muscle cells (Chow et al.,
2009) which may impact on the anti-inflammatory activity of
ghrelin and its involvement in atherosclerosis (Waseem et al.,
2008). In ghrelin receptor-expressing HEK293 cells, the domi-
nant-negative activity of GHS-R1b results in decreased constitutive
activation of phosphatidylinositol-specific phospholipase C (PI-
PLC) but does not affect ghrelin receptor signalling to extracellular
signal-regulated kinases 1/2 (ERK1/2) (Chu et al., 2007; Leung
et al., 2007). These results suggested that any up-regulation of
GHS-R1b expression might preferentially attenuate functional
activity dependent on the constitutive activity of ghrelin receptors,
while leaving agonist-stimulated ghrelin receptor signalling unaf-
fected. However, the full functional consequences of GHS-R1b over
expression relative to GHS-R1a remain to be determined.
In co-transfection assays, specific GHS-R1a/GHS-R1b heterodi-
mers were detected by bioluminescence resonance energy transfer
(BRET) and immunoprecipitation assays (Leung et al., 2007). The
formation of homodimers and heterodimers results in the produc-
tion of receptor complexes with altered trafficking, signalling, and
pharmacological properties (Salahpour et al., 2000; Angers et al.,
2002; Milligan, 2004; Maggio et al., 2005; Milligan, 2010). Indeed,
GHS-R1b heterodimerizes with the neurotensin receptor (NSR1) to
generate a receptor responsive to neuromedin U (Takahashi et al.,
2006). Our original studies confirmed those of Smith et al. (2005)
indicating that ghrelin receptors were localized to the plasma
membrane and cytoplasm of HEK293 cells transiently expressing
ghrelin receptors, while GHS-R1b was in an intracellular compart-
ment. Furthermore, the BRET50 values for GHS-R1a homodimers
and GHS-R1a/GHS-R1b heterodimers were similar, indicating a
similar probability of GHS-R1a forming homo- or hetero-dimers
when expressed in the same cell as GHS-R1b (Leung et al., 2007).
Appropriate dimer/oligomer formation appears to be essential for
cell surface delivery of class A and class C GPCRs (Milligan,
2010), therefore, the dominant-negative activity of GHS-R1b might
best be explained by the formation of GHS-R1a/GHS-R1b heterodi-
mers within the endoplasmic reticulum (ER). The purpose of the
current study was therefore to identify the subcellular localization
of ghrelin receptors in the presence and absence of GHS-R1b, and
to determine if the dominant-negative effect of GHS-R1b was in-
deed due to heterodimerization within the ER.
2. Materials and methods
2.1. Materials
Human GHS-R1 and human GHS-R1b cDNA were purchased
from Guthrie Research Institute (Rolla, MD, USA), and the CD8-
Rluc cDNA construct was a gift from Prof. M. Bouvier (Montreal
University, Canada). Cell culture reagents, including Lipofect-
AMINE 2000 reagents, were purchased from Invitrogen (Carlsbad,
CA, USA). Hybond nitrocellulose filters were from Pall Life Sciences
(Pensacola, FL, USA) and the ECL kit was from GE Medical Systems
Hong Kong Ltd. Micro BCA Protein Assay Kit was from Pierce
(Rockford, IL, USA). The codon humanized pRenilla luciferase-N
vector (pRluc-N2) and the codon humanized green fluorescent
protein vector (pGFP2-N) were purchased from PerkinElmer
(Atlanta, GA, USA). Antibody sources were: a-6F [Na+/K+ ATPase]
(Developmental Studies Hybridoma Bank, University of Iowa,
USA); calnexin (StressGen, Ann Arbor, MI, USA); GHS-R1a and
GHS-R1b (Phoenix Biotech (Beijing) Co. Ltd.); phospho-ERK1/2,
ERK1/2 and horseradish peroxidase-linked goat anti-rabbit immu-
noglobulin (Cell Signaling, Danvers, MA, USA). CellMask Deep Red
Plasma Membrane stain, BODIPY Golgi TR C5-ceramide stain and
ER Tracker Red stain were purchased from Invitrogen (Carlsbad,
CA, USA). All other compounds were supplied by Invitrogen (Carls-
bad, CA, USA) or Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Cell culture and transfection reagents
HEK293 cells were purchased from ATCC and were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) containing
100 i.u./ml penicillin, 100 lg/ml streptomycin and 10% foetal bo-
vine serum, and were incubated in a humidified atmosphere of
5% CO2/95% air at 37 °C. HEK293 cells were transfected at 80% con-
fluency using LipofectAMINE 2000 liposome reagent and Opti-
mem I reduced serum medium for 5 h, according to the manufac-
turer’s instructions. All cells were harvested 48 h post-transfection.
The cDNA constructs (GHS-R1a-Rluc, 2myc-GHS-R1a-GFP2, 3HA-
GHS-R1b, GHS-R1b-Rluc, 3HA-GHS-R1b-GFP2 and CD8-Rluc) were
prepared as described previously (Leung et al., 2007; Chow et al.,
2008). HEK293 cells were co-transfected with Rluc and GFP2 con-
structs at a maximal cDNA concentration of 1 lg/ml and at a cDNA
ratio of 1:5 (0.167 lg/ml Rluc-construct + 0.833 lg/ml GFP2 con-
struct), to generate a near maximal BRET2 signal.
2.3. Immunocytochemistry
HEK293 cells were seeded at a cell density of 1.5  105 cells/
well on poly-D-lysine-coated 22 mm glass coverslips in 6-well tis-
sue culture plates. For live cell imaging, cells were stained with
various organelle dyes (CellMask Deep Red Plasma Membrane
stain, BODIPY Golgi TR C5-ceramide stain or ER Tracker Red stain)
according to the manufacturer’s instructions, and observed using a
Leica SP5 laser scanning confocal microscope equipped with Ar-
gon/DPSS/HeNe lasers (Leica Microsystems GMSGmbH, Germany).
GFP2 was excited at 476 and 488 nm with a laser power ratio of
2:1, and fluorescence was captured at 525 ± 25 nm. The captured
images were processed for publication using Adobe Photoshop
CS4 (Adobe Systems, CA, USA).
2.4. Subcellular fractionation
According to the methods described by Guan et al. (2009), cells
were rinsed with PBS, harvested using a cell scraper, and centri-
fuged at 300g for 5 min. A 10-fold volume of ice-cold hypotonic ly-
sis buffer (20 mM Hepes, pH 7.4; 2 mM EDTA; 2 mM EGTA; 6 mM
MgCl2) with protease inhibitors (Complete Tablets, EDTA-free;
Roche Applied Science, Indianapolis, IN, USA) was added to the
resulting pellet and the cells were allowed to swell for 10 min at
4 °C. Cells were homogenized with 15 strokes of a Dounce homog-
enizer then centrifuged at 1000g for 10 min at 4 °C. The post-nucle-
ar supernatant (fraction S; approximately 1–3 mg protein) was
collected, resuspended in 2 M sucrose, and placed in the bottom
of a 12 ml ultracentrifuge tube for a Beckman SW41 Ti rotor, and
overlayed with 1.9 M, 1.75 M, 1.5 M, 1.25 M, 1.0 M, 0.75 M and
0.5 M sucrose. The discontinuous step sucrose gradient was centri-
fuged at 207,000g for 16 h at 4 °C. Aliquots of 1 ml were taken from
the top of each tube and BRET2 ratios were determined in duplicate
100 ll samples. To follow the distribution of Rluc-tagged proteins,
luciferase luminescence was also determined in duplicate 100 ll
samples, using colenterazine H (5 lM) as described previously
(Leung et al., 2007). Equal volume samples were also taken from
each aliquot for Western blotting where the expression of Na+/K+
ATPase (a marker for the plasma membrane) and calnexin (a mar-
ker for the endoplasmic reticulum) was expressed relative to that
 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254
in fraction S run on the same gel. Protein content of each sample
was determined using Micro BCA Protein Assay Kit.
2.5. BRET2 assay
Bioluminescence resonance energy transfer (BRET) assays are
proximity-based assays which can provide a quantitative analysis
of receptor dimerization in living cells (Mercier et al., 2002). Because
BRET signals arise from the close interaction between Rluc- and
GFP2-expressing proteins, it is essential to distinguish specific and
non-specific BRET signals (Mercier et al., 2002; Ayoub and Pfleger,
2010). We have previously shown that HEK293 cells expressing
GHS-R1a/GHS-R1a or GHS-R1a/GHS-R1b displayed BRET2 satura-
tion curves (Leung et al., 2007), indicative of a specific interaction
between GHS-R1a homodimers and GHS-R1a/GHS-R1b heterodi-
mers. In contrast, the interactions between GHS-R1b/GHS-R1b or
CD8/GHS-R1a were non-specific in character because they showed
quasi-linear BRET2 saturation curves (Leung et al., 2007; Chow
et al., 2008). Therefore, to demonstrate possible non-specific BRET2
in the subcellular fractions, we again used a CD8-Rluc cDNA which
consists of a truncated form of the integrin CD8 fused to Rluc which
is reported to exhibit a subcellular distribution similar to that of the
GPCRs, but is not expected to interact with them (Galés et al., 2005).
Samples (100 ll) from each of the 12 sucrose gradient fractions
were transferred to two wells of a 96-well microplate (white Opti-
plate, PerkinElmer, Atlanta, GA, USA). The Rluc substrate Deep-
BlueCTM (PerkinElmer, Atlanta, GA, USA) was added to a final
concentration of 5 lM, and readings were collected immediatedly
using a FUSION microplate analyser (Packard, Meriden, CT, USA)
that allows integration of signals detected at 410 nm and
515 nm, and normalized BRET2 ratios were determined as de-
scribed previously (Leung et al., 2007). The BRET2 ratio is defined
as the BRET2 ratio for the co-expression of the Rluc and GFP2 con-
structs normalized against the BRET2 ratio for the Rluc expression
construct alone. The normalized BRET2 ratio is defined as (emission
at 515 nm of co-transfected cells minus emission at 515 nm of
untransfected cells)/(emission at 410 nm of co-transfected cells
minus emission at 410 nm of untransfected cells) – cf, where cf
corresponds to (emission at 515 nm of Rluc-expressing cells minus
emission at 515 nm of untransfected cells)/(emission at 410 nm of
Rluc-expressing cells minus emission at 410 nm of untransfected
cells) for the Rluc construct expressed alone in the same experi-
ment. Assays were performed in duplicate at room temperature.
2.6. Western blotting
The fractions isolated from the sucrose density gradient (25 ll)
were mixed with sample loading buffer, separated by 10%
SDS–polyacrylamide gel electrophoresis, and electrophoretically
transferred onto Hybond nitrocellulose membranes. Membranes
were probed with anti- a-6F for plasma membrane (Na+/K+ ATP-
ase; 1:1000) and calnexin for endoplasmic reticulum (1:2000).
Phospho-ERK1/2 (1:500) and total ERK1/2 (1:1000) were sequen-
tially detected by specific primary antisera and horseradish perox-
idase-linked goat anti-rabbit immunoglobulin. The immunoblots
were visualized by chemiluminescence with an enhanced chemilu-
minescence (ECL) kit, and the band intensities were quantified
using the computer program Image J (NIH, USA).
2.7. Data analysis
Values reported are means ± SEM. Comparisons between groups
were made using ANOVA (analysis of variance) with Bonferroni’s
post hoc tests, as appropriate, using GraphPad Prism software ver-
sion 5.0 (GraphPad Software Inc., San Diego, USA). Statistical signif-
icance was taken as P < 0.05.
249
3. Results
3.1. The subcellular localization of GHS-R1a ± GHS-R1b in whole cells
We were unable to unambiguously determine the localization
of ghrelin receptors and GHS-R1b in native cells due to the lack
of specificity of commercially available antibodies: anti-GHS-R1a
and anti-GHS-R1b antibodies both showed immunoreactivity in
pcDNA3.1-transfected HEK293 cells which should be devoid of
these proteins (see Lau et al., 2009) and both antibodies cross-re-
acted in cell lysates run on Western blots (data not shown). There-
fore, although over-expression systems may not completely reflect
the situation in native cells, they are a useful starting point for
these investigative studies. The subcellular localization of these
proteins was therefore determined using live cell microscopy in
HEK293 cells transiently transfected with 2myc-GHS-R1a-GFP2 or
3HA-GHS-R1b-GFP2 (Fig. 1). We made use of the GFP2 tags to de-
tect the GHS-R constructs and cell stains for intracellular organ-
elles. The overall distribution of 2myc-GHS-R1a-GFP2 and 3HA-
GHS-R1b-GFP2, as detected from GFP fluorescence, was similar to
that of 2myc-GHS-R1a and 3HA-GHS-R1b, as detected by the C-ter-
minal HA and myc tags (data not shown). 2myc-GHS-R1a-GFP2 co-
localized with the plasma membrane (CellMask Deep Red), the
Golgi apparatus (BODIPY TR C5-ceramide) and the ER (ER Tracker
Red) (Fig. 1A). In contrast, 3HA-GHS-R1b-GFP2 co-localized with
the ER and Golgi apparatus markers but was absent from the plas-
ma membrane (Fig. 1B). Next we examined the effect of co-trans-
fecting HEK293 cells with 2myc-GHS-R1a-GFP2 and 3HA-GHS-R1b,
at cDNA ratios of 1:5 to match the optimum conditions for the
BRET2 assay for receptor dimerization (Leung et al., 2007). In the
presence of 3HA-GHS-R1b, the expression of 2myc-GHS-R1a-
GFP2 in the plasma membrane and Golgi apparatus was decreased,
while still abundantly expressed in the ER (Fig. 1C).
3.2. The localization of GHS-R1a and GHS-R1b in subfractioned cells
To determine the subcellular localization of GHS-R1a/GHS-R1b
heterodimers, we first prepared subcellular fractions of transfected
HEK293 cells according to Guan et al. (2009). With this method,
the HEK293 cell membrane fraction was applied to the bottom of
a discontinuous sucrose density gradient, and fractions were re-
solved by flotation. From the distribution of markers, as assayed
by Western blotting, the plasma membrane fractions were repre-
sented by fractions 1–5, and the ER fractions were in 5–9 (Fig. 2C).
The distribution of receptor-Rluc constructs was determined by
monitoring luciferase activity in the sucrose gradient fractions. We
identified two peaks of GHS-R1a-Rluc expression, one in plasma
membrane fractions and a second peak of luciferase activity in
the ER fractions (Fig. 2A). This method of analysis did not reveal
any significant change in the distribution of GHS-R1a-Rluc when
HEK293 cells were co-transfected with 3HA-GHS-R1b-GFP2
(Fig. 2A). 3HA-GHS-R1b-Rluc alone was not detected in the plasma
membrane fractions and showed a similar ER distribution profile as
CD8-Rluc (Fig. 2B) which was quite distinct from that of GHS-R1a-
Rluc (Fig. 2A).
3.3. The localization of GHS-R1a homodimers and GHS-R1a/GHS-R1b
heterodimers in subfractionated cells
To determine the precise subcellular localization of BRET2 sig-
nals, BRET2 was assayed in all 12 fractions using Rluc- and GFP2-
tagged receptor constructs. Raw data for fractions 1–9 is presented
and shows a steady decline in BRET2 from GHS-R1a homodimers
(Fig. 3A) and a slight increase in BRET2 in the ER fractions for GHS-
R1a/GHS-R1b heterodimers (Fig. 3A). BRET2 signals in the bottom
250 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254

Fig. 1. Ghrelin receptors (GHS-R1a) and the mutant ghrelin receptor polypeptide (GHS-R1b) show different subcellular localization patterns and GHS-R1b traps GHS-R1a in
the ER. HEK293 cells were transfected with GFP2-tagged receptors (green) and stained with subcellular markers (red) of the plasma membrane (CellMask Deep Red), the Golgi
apparatus (BODIPY TR C5-ceramide) and the ER (ER Tracker Red). Live cell confocal microscopy images are shown which are representative of three independent experiments.
(A) 2myc-GHS-R1a-GFP2 co-localized with markers of plasma membrane, Golgi and ER; (B) 3HA-GHS-R1b-GFP2 was only detected in the ER; and (C) less 2myc-GHS-R1a-GFP2
was observed in the plasma membrane and Golgi compartments in cells co-expressing 3HA-GHS-R1b. Images Scale bars = 10 lm. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this paper.)

Fig. 2. The expression of Rluc receptor constructs in the sucrose density gradient. HEK293 cell membrane lysates were fractionated as described in Section 2, and Rluc activity
was assayed in duplicate 100 ll fractions. (A) GHS-R1a-Rluc shows a two-phase distribution profile in the absence (s) and presence of 3HA-GHS-R1b-GFP2 (d), with the
major peak in fraction 4 and a second around fraction 7. (B) Both GHS-R1b-Rluc (h) and CD8-Rluc (j) show a single-phase distribution pattern, peaking in fraction 7. Data are
means ± SEM, from 4 independent experiments, with each sample assayed in duplicate. (C) Equal volume fractions were analysed by Western blotting for Na+/K+ ATPase (d;
plasma membrane marker) and calnexin (N; ER marker), and a representative blot from 4 independent experiments is shown.
 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254 251

fractions have been excluded as these were extremely variable. In
living cell BRET2 assays, bystander BRET2 can arise from random col-
lisions of Rluc- and GFP2-tagged receptors promoted by a high
receptor density (Mercier et al., 2002). It is possible that bystander
BRET2 might also arise from aggregation of proteins in these bottom
fractions of the sucrose density gradient. Because absolute BRET2
values cannot be used as a quantitative measure of the relative
number of homo/heterodimers formed for the different combina-
tions (Ayoub and Pfleger, 2010), we normalized the BRET2 data to
the signal from fraction 4 for each pair of BRET2 constructs. This
method allowed us to better identify any relative change in the
localization of the GHS-R1a homodimers compared with GHS-R1b
homodimers, and with GHS-R1a/GHS-R1b and CD8/GHS-R1a het-
erodimers. This method of analysis resulted in very high variability
in fractions 1 and 2, but it is evident that specific GHS-R1a homodi-
mers appear evenly distributed in plasma membrane and ER frac-
tions (Fig. 3C). The BRET2 signals from cells expressing GHS-R1b/
GHS-R1b or CD8/GHS-R1a are non-specific in character because
they show quasi-linear BRET2 saturation curves (Leung et al.,
2007; Chow et al., 2008), and here they showed no remarkable pat-
tern of distribution (Fig. 3C,D). In marked contrast, the BRET2 signals
from specific GHS-R1a/GHS-R1b heterodimers were significantly
increased in the ER compared with BRET2 from GHS-R1a homodi-
mers (Fig. 3B).
3.4. Activation of ERK1/2 by GHS-R1a in the endoplasmic reticulum
Constitutive activation of ERK1/2 by ghrelin receptor-express-
ing cells was readily detected by Western blotting of cell lysates,
especially in fractions 7–10 (Fig. 4). Protein recovery in fractions
1–5 was insufficient to detect tERK1/2, but peaked at approxi-
mately 50 lg/100 ll in fraction 8. Therefore, the lack of data from
fractions 1–6 does not necessarily mean lack of activation of ERK1/
2 by GHS-R1a in these fractions, but any ERK1/2 activity was below
the level of detection. To account for different protein loading on
the gels, estimates of ERK1/2 activity were determined from the ra-
tio of pERK1/2 to tERK1/2 detected for each fraction. We observed a
statistically significant increase in ERK1/2 activity in the ER frac-
tions of cells expressing GHS-R1a when compared with cells trans-
fected with the vector control (P < 0.05; two-way ANOVA), and this
effect remained in cells co-expressing GHS-R1a and GHS-R1b
(Fig. 4).
4. Discussion
In the present study, HEK293 cells expressed the ghrelin recep-
tor in the plasma membrane, Golgi and ER fractions, which is ex-
pected of a GPCR in heterologous expression systems, and in
native cells and tissues (Gobeil et al., 2006). The ghrelin receptor
possesses a putative nuclear recognition sequence which would al-
low for binding to importins and targeting of the protein to the nu-
cleus (Christophe et al., 2000), however the present study failed to
find any evidence of nuclear translocation of ghrelin receptors even
in the presence of GHS-R1b. Contrary to ours (Leung et al., 2007)
and other’s work (Smith et al., 2005), the confocal microscopy
images in the present study clearly show that GHS-R1b is not ex-
pressed in the nucleus. Indeed, the intracellular localization of
GHS-R1b observed previously using ELISA-based assays (Leung
et al., 2007) appears due to concentration of GHS-R1b within the
ER compartment. The ER forms part of the cellular quality control
machinery where functionally inactive mutant GPCRs can be pre-
vented from expression at the cell surface (Salahpour et al.,
2004). We have previously shown that GHS-R1b acts as a domi-
nant-negative mutant of the ghrelin receptor and decreases its cell
surface expression (Leung et al., 2007). In the current study, both
2myc-GHS-R1a-GFP2 and 3HA-GHS-R1b-GFP2 appeared in the ER,

Fig. 3. GHS-R1a/GHS-R1b heterodimers are concentrated in the ER fractions. HEK293 cell membrane lysates were fractionated as described in Section 2, with fraction 1–5
representing the plasma membrane fractions and 5–9 representing the ER fractions. The absolute BRET2 ratios for GHS-R1a-Rluc + 2myc-GHS-R1a-GFP2 (d) and GHS-R1a-
Rluc + 3HA-GHS-R1b-GFP2 (s) are shown in fractions 1–9 (A). The BRET2 ratios have been normalized to the BRET2 value in fraction 4, to allow comparison between different
BRET pairs: (B) GHS-R1a-Rluc + 2myc-GHS-R1a-GFP2 (d) and GHS-R1a-Rluc + 3HA-GHS-R1b-GFP2 (s); (C) GHS-R1a-Rluc + 2myc-GHS-R1a-GFP2 (d) and GHS-R1b-
Rluc + 3HA-GHS-R1b-GFP2 (4); (D) GHS-R1a-Rluc + 2myc-GHS-R1a-GFP2 (d) and CD8-Rluc + 2myc-GHS-R1a-GFP2 (N). Data are means ± SEM, from 4 independent
experiments, with each sample assayed in duplicate. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 compared with control cells expressing GHS-R1a-Rluc + 2myc-GHS-R1a-GFP2; 2-way
ANOVA.
252 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254
Fig. 4. The presence of GHS-R1b does not affect the constitutive activation of ERK1/
2 by GHS-R1a in the ER. HEK293 cell membrane lysates were fractionated and the
ratio of pERK1/2 and tERK1/2 expression was determined as described in Materials
and Methods. Representative blots are shown in (B), (C) and (D). (A) Endogenous
ERK1/2 activity was very low in control cells transfected with empty vector
(pcDNA3.1; h), but significantly increased in cells expressing GHS-R1a-Rluc (d);
⁄ Using a method of quantitative BRET analysis (Mercier et al.,
P < 0.05 compared with control cells expressing pcDNA3.1 (2-way ANOVA). The
presence of 3HA-GHS-R1b-GFP2 did not affect the activity of GHS-R1a-Rluc (s).
Data are means ± SEM, of 3 independent experiments.
and the presence of GHS-R1b appeared to diminish co-localization
of GHS-R1a with plasma membrane and Golgi markers. Our results
suggest that GHS-R1b might decrease the cell surface expression of
ghrelin receptors by hindering their progression from the ER to the
Golgi apparatus, and thus decreasing their ultimate transport to
the plasma membrane.
GPCR mutants frequently exert a dominant-negative effect on
the cell signalling activity of corresponding full length receptors,
as has been shown for the a1A-adrenergic receptor (Cogé et al.,
1999), a2-adrenergic receptors (Zhou et al., 2006), chemokine
CCR5 and CXCR2 receptors (Benkirane et al., 1997; Trettel et al.,
2003), dopamine D3 receptor (Karpa et al., 2000; Elmhurst et al.,
2000), gonadotrophin receptors (Grosse et al., 1997), histamine
H4 receptors (van Rijn et al., 2008), prostacyclin receptors (Ibrahim
et al., 2010), prostaglandin E2 EP1 receptor subtype (Okuda-Ashi-
taka et al., 1997), relaxin receptors (Kern et al., 2008), secretin
receptors (Ding et al., 2002) and the V2 vasopressin receptor
(Zhu and Wess, 1998). These dominant-negative mutants can re-
sult in physiological outcomes in humans, such as red hair colour
and skin cancer for loss-of-function melanocortin 1 receptors
(MC1R) (Sánchez-Laorden et al., 2009), and accelerated cardiovas-
cular disease in the case of prostacyclin receptors (IPR212C) (Ibrahim
et al., 2010). In many cases, these mutant GPCRs are proposed to
heterodimerize with the full length GPCRs within the ER, support-
ing the view that homodimerization is a prerequisite for receptor
trafficking to the cell surface (Lohse, 2010; Milligan, 2010).
To assess this hypothesis as the mechanism underlying the dom-
inant-negative activity of GHS-R1b (Leung et al., 2007), we used a
sucrose gradient centrifugation method to fractionate membranes
isolated from cells co-expressing Rluc- and GFP2-tagged receptor
constructs in order to determine if the subcellular localization of
ghrelin receptors was affected by the formation of specific hetero-
dimers with GHS-R1b. By following the expression of Rluc-tagged
proteins, we have shown that ghrelin receptors were localized both
in the plasma membrane (those fractions expressing the plasma
membrane marker Na+/K+ ATPase) and the ER (those fractions
expressing the ER marker calnexin), matching a similar subcellular
distribution pattern reported for b2-adrenergic receptors (Salah-
pour et al., 2004). In contrast to the ghrelin receptor, GHS-R1b
was not present in the plasma membrane and localized in the ER
fractions in a similar manner to the integrin CD8. The CD8-Rluc con-
struct used here as the negative control in the BRET2 assays (Chow
et al., 2008) was reported to exhibit a subcellular distribution pro-
file similar to that of GPCRs when visualized by confocal immuno-
fluorescence microscopy (Galés et al., 2005). Nevertheless, in the
present study, CD8-Rluc remained in the ER with a similar subcel-
lular distribution profile as GHS-R1b. Importantly, the presence of
GHS-R1b did not significantly change the distribution profile of
ghrelin receptors in these subcellular fractions. These observations
match those of Leung et al. (2007) where receptor ELISA assays indi-
cated that, despite the decreased cell surface expression of ghrelin
receptors, their relative distribution in extracellular and intracellu-
lar compartments remained similar in the absence and presence of
GHS-R1b. Monitoring the influence of GHS-R1b on the absolute
expression of ghrelin receptors cannot readily be performed by
detecting only Rluc-tagged ghrelin receptors in this experiment. Be-
cause the control HEK293 cells were also transfected with a 5-fold
excess of 2myc-GHS-R1a-GFP2 cDNA, then GHS-R1a-Rluc will make
up less than 17% of total ghrelin receptor expression and any de-
crease in plasma membrane expression of GHS-R1a-Rluc will be
hard to detect with this methodology.
2002), we determined previously that the likelihood of forming
GHS-R1a/GHS-R1a homodimers and GHS-R1a/GHS-R1b heterodi-
mers was similar (Leung et al., 2007). Therefore, any ghrelin recep-
tors within the ER compartment would have to compete with GHS-
R1b for the formation of GHS-R1a homodimers for trafficking to
the cell surface or in the formation of GHS-R1a/GHS-R1b heterodi-
mers which would be retained in this intracellular compartment.
The BRET2 signal from GHS-R1a/GHS-R1a homodimers followed a
similar distribution profile between plasma membrane and ER
fractions as described for b2-adrenergic receptors (Salahpour
et al., 2004) and lutropin receptors (Guan et al., 2009), being more
evenly distributed throughout the subcellular fractions. This is be-
cause BRET is a ratiometric value (Salahpour et al., 2004), so the
proportion of GHS-R1a-Rluc interacting with 2myc-GHS-R1a-
GFP2 was similar in all the subcellular fractions.
As expected from our previous work (Leung et al., 2007), the
BRET2 signal from HEK293 cell membranes expressing GHS-R1a/
GHS-R1a homodimers was much greater than from cells express-
ing GHS-R1a/GHS-R1b heterodimers. This means that the effi-
ciency of resonance energy transfer between the energy donor
(GHS-R1a-Rluc) and acceptor (2myc-GHS-R1a-GFP2) was greater
than between GHS-R1a-Rluc and 3HA-GHS-R1b-GFP2, but does
not necessarily mean that there were more GHS-R1a homodimers
than GHS-R1a/GHS-R1b heterodimers. To account for the differ-
ences in BRET efficiencies, we normalized the BRET2 signals for
individual donor/acceptor pairs, and found that the proportion of
GHS-R1a/GHS-R1b heterodimers was increased within the ER frac-
tions, whereas no obvious changes were observed for GHS-R1a
 K.B.S. Chow et al. / Molecular and Cellular Endocrinology 348 (2012) 247–254
homodimers. Interpretation of BRET assays is prone to error when
using single concentrations of Rluc and GFP2 constructs (James
et al., 2006), but we have previously used BRET2 saturation analysis
and negative controls to study ghrelin receptor dimerization in
whole cells (Leung et al., 2007; Chow et al., 2008). Thus, in
HEK293 cells co-transfected with GHS-R1b-Rluc and 3HA-GHS-
R1b-GFP2 or with CD8-Rluc and 2myc-GHS-R1a-GFP2, BRET2 satu-
ration curves were quasi-linear (Leung et al., 2007), indicating that
the BRET2 signals resulted from random collision events (Mercier
et al., 2002; Ayoub and Pfleger, 2010). In the current study, non-
specific GHS-R1b homodimers and non-specific CD8/GHS-R1a het-
erodimers were not concentrated in the ER fractions. Together,
these data support our hypothesis that GHS-R1b retains ghrelin
receptors within the ER through the specific process of heterodi-
merization, and this could account for the decreased cell surface
expression of ghrelin receptors in the presence of GHS-R1b. De-
spite the formation of specific GHS-R1a/GHS-R1b heterodimers in
the ER, sufficient GHS-R1a/GHS-R1a homodimers must be inserted
in the plasma membrane to respond to ghrelin for stimulation of
phospholipase C and activation of ERK1/2 assays (Chu et al.,
2007; Leung et al., 2007). Thus, high expression levels of ghrelin
receptors are not essential for maximal stimulation of these cell
signalling pathways.
Ghrelin receptor signalling through Gq-proteins occurs predom-
inantly at the plasma membrane (Holliday et al., 2007). Although
ghrelin-stimulated ERK1/2 activity was also dependent on G-pro-
tein signalling in whole cells (Camina et al., 2007; Chu et al.,
2007), the precise localization of this activity was unknown. Nev-
ertheless, the cell signalling activity of GPCRs is not necessarily
limited to the plasma membrane, with persistent signalling occur-
ring in a G-protein-dependent manner from GPCRs internalized in
endosomes (Calebiro et al., 2010). In whole cells transiently
expressing ghrelin receptors, ghrelin-stimulated ERK1/2 activity
was unaffected by coexpression with GHS-R1b, and data suggested
that ERK1/2 activation did not depend on constitutive ghrelin
receptor activity (Chu et al., 2007). In the current study, we have
directly compared basal (constitutive) ghrelin receptor activity
with ERK1/2 activation in HEK293 cells expressing empty vector
(pcDNA3.1), and have detected a small, but statistically significant,
increase in pERK/tERK in the ER fractions of ghrelin receptor-
expressing cells which was unaltered by co-expression with
GHS-R1b. In HEK293 cells expressing GHS-R1a homodimers, ago-
nists can decrease BRET2 but this effect is lost in the presence of
GHS-R1b (Leung et al., 2007). As the chance for forming GHS-
R1a/GHS-R1a homodimers and GHS-R1a/GHS-R1b heterodimers
is similar, it is likely that constitutive activation of ERK1/2 involves
GHS-R1a/GHS-R1a homodimers.
Because ghrelin-stimulated ERK1/2 was independent of ghrelin
receptor internalization (Chu et al., 2007), the detection of pERK1/2
in subcellular fractions may not be dependent on prior activation
of cell surface ghrelin receptors and their subsequent internaliza-
tion. Indeed, intracellularly-retained GPCRS, such as the Gs-cou-
pled vasopressin receptor (V2R) localized in the ER, can still
respond to cell permeable agonists (Robben et al., 2009). The cellu-
lar microenvironment will determine which substrates are prefer-
entially phosphorylated by activated ERK1/2 in cells expressing
ghrelin receptors (Casar et al., 2009). As the ER is contiguous with
the nuclear membrane (Du et al., 2004), it is conceivable that ghre-
lin receptors in the ER may be functionally active. At this time
however, the organizational features and spatial orientation of
ghrelin receptor homodimers in the ER remains to be determined.
Nuclear glutamate receptors are orientated with their ligand bind-
ing domain inside the nucleoplasmic reticulum, and glutamate can
access their binding sites by active transport into this subcellular
compartment (Kumar et al., 2008). Because ghrelin receptors are
constitutively active, ligand access into ER membranes is not
253
essential for receptor signalling, but it remains to be determined
if the relatively small changes observed here are physiologically
relevant.
In conclusion, these results suggest that ghrelin receptors can
be retained in the endoplasmic reticulum by heterodimerization
with GHS-R1b, and constitutive activation of phospholipase C is
attenuated due to decreased cell surface expression of ghrelin
receptors. However, in the presence of GHS-R1b, sufficient ghrelin
receptor homodimers can still be expressed on the cell surface for
maximal responses to agonist stimulation.
Acknowledgements
This work was fully supported by a Grant from the Research
Grants Council of the Hong Kong Special Administrative Region
(GRF475807). The authors would like to thank Dr. Deborah
L. Segaloff for invaluable advice on the preparation and use of
the sucrose density gradient, and Prof. Micheal Bouvier for the
CD8-Rluc construct.
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