THE GHRELIN RECEPTOR ISOFORMS (GHS-R1a AND GHS-R1b) AND GPR39: AN INVESTIGATION INTO RECEPTOR DIMERISATION Peter Stephen Cunningham Bachelor of Applied Science (Hons) Institute of Health and Biomedical Innovation School of Life Sciences, Queensland University of Technology A thesis submitted for the degree of Doctor of Philosophy of the Queensland University of Technology 2010 AGE
236
Embed
THE GHRELIN RECEPTOR ISOFORMS (GHS-R1a AND ...eprints.qut.edu.au/39443/1/Peter_Cunningham_Thesis.pdfR1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
i
THE GHRELIN RECEPTOR ISOFORMS
(GHS-R1a AND GHS-R1b) AND GPR39:
AN INVESTIGATION INTO RECEPTOR
DIMERISATION
Peter Stephen Cunningham
Bachelor of Applied Science (Hons)
Institute of Health and Biomedical Innovation
School of Life Sciences, Queensland University of Technology
A thesis submitted for the degree of Doctor of Philosophy of
and Korolev 1996). Tunicamycin has previously been used to induce apoptosis and
to illustrate the protective effect of GPR39 in a mouse hippocampal cell line and in
HEK293 cells (Dittmer et al. 2008). Cells were transfected with an empty vector
control, or vectors containing GHS-R1a, GPR39 alone or GHS-R1a and GPR39 in
combination for 6 hr, followed by the measurement of apoptosis after 24 hr treatment
with 2 µg/ml tunicamycin (Figure 6.3). Treatment with tunicamycin significantly
increased cell apoptosis in all cell combinations (p<0.01). Transfection with GHS-
R1a and/or GPR39, however, did not produce a protective effect when compared to
cells similarly transfected with an empty vector control and treated with tunicamycin
(Figure 6.3).
160
Figure 6.3 Overexpression of GHS-R1a and GPR39 alone, or in combination,
did not attenuate apoptosis induced by tunicamycin in PC-3 prostate cancer
cells. Cells were transfected and after 6 hr were treated with 2 μg/ml tunicamycin for
24 hr to induce endoplasmic reticulum stress-related apoptosis. The level of
apoptosis in transfected and treated cells was normalised to the level of apoptosis in
the vehicle control treated sample (empty vector transfected cells) to give the relative
enrichment factor of DNA fragments. In all cases, apoptosis was significantly
increased when compared to the vehicle control in tunicamycin treated cells
(p<0.01). No significant change in tunicamycin-induced apoptosis was observed in
cells overexpressing GHS-R1a or GPR39 alone, or in combination, when compared
to cells transfected with an empty vector control. Data represent the mean ± SD of
duplicate measurements. Statistical analysis was performed by one way ANOVA
with Tukey’s post-hoc test for comparisons of all means.
161
The potential role of the ERK1/2 signalling pathway in PC-3 cell survival in cells
overexpressing GHS-R1a alone, or in combination with GHS-R1b or GPR39, was
also examined. Activation of the ERK1/2 pathway promotes cell survival and
treatment with the specific MEK (mitogen-activated protein kinase kinase, which
activates ERK1/2) inhibitor, U0126, induces cell apoptosis in normal and cancerous
cell lines (Shakibaei et al. 2001; Blank et al. 2002; Huynh et al. 2003; Rice et al.
2004; Rice et al. 2006). PC-3 prostate cancer cells were treated with U0126 to induce
apoptosis. Cells were additionally treated with ghrelin, obestatin and zinc to asses if
any cell survival effect on cells overexpressing the receptors could be affected by
ligand treatment. The ERK1/2 signalling pathway plays a role in the ghrelin
mediated cell survival (Baldanzi et al. 2002; Kim et al. 2004; Mazzocchi et al. 2004;
Chung et al. 2007; Granata et al. 2007; Zhang et al. 2007b).
PC-3 cells were transfected with an empty vector control, or vectors encoding GHS-
R1a, alone or in combination with GHS-R1b or GPR39 vector constructs.
Transfected cells were treated with a ligand (10 nM ghrelin, 10 nM obestatin, 10 µM
zinc or vehicle control) in addition to the specific inhibitor of MEK, U0126 (10 µM),
or a vehicle control for 48 hrs. Apoptosis was measured as previously described
(Figure 6.4). As phosphorylation of ERK1/2 promotes cell survival, it may be
expected that an ERK1/2 inhibitor would result in increased basal apoptosis,
independent of GPCR transfection, however, this was not observed. Unexpectedly,
treatment with U0126 did not result in any significant change in apoptosis in any
transfected and treated cells.
162
Figure 6.4 The MEK inhibitor, U0126, did not stimulate an increase in
apoptosis in PC-3 cells overexpressing GHS-R1a alone, or in combination with
GHS-R1b, or GPR39 and treated with ghrelin, obestatin or zinc. The level of
apoptosis in transfected and treated cells was normalised to the level of apoptosis in
the vehicle control treated sample (empty vector transfected cells) to give the relative
enrichment factor of DNA fragments. PC-3 cells were treated with 10 µM U0126 to
induce apoptosis, however, no significant change in basal apoptosis was observed.
Data for the vehicle control and ghrelin treated samples are from three independent
experiments performed in duplicate. Data for the obestatin and zinc treated samples
are from two independent experiments performed in duplicate. Mean ± SEM.
Statistical analysis was performed by one way ANOVA.
163
6.4 DISCUSSION
In this study we investigated the effects of GHS-R1a, GHS-R1b and GPR39, alone
and in combination on ERK1/2 and AKT signalling and apoptosis. Our studies have
provided conflicting results concerning the dimerisation of GHS-R1a, GHS-R1b and
GPR39, receptors within the ghrelin receptor family. Our co-immunoprecipitation
experiments demonstrated that heterodimers could form between GHS-R1a, GHS-
R1b and GPR39, however, using resonance energy transfer techniques, we were
unable to confirm or exclude the formation of specific dimers between these
receptors. The demonstration of specific functional outcomes from receptor
dimerisation is an important step in demonstrating the physiological significance of
receptor interactions. Interactions between a number of GPCRs have led to altered
functional outcomes, such as altered binding affinity, signal transduction and
receptor internalisation (Satake and Sakai 2008) and some functional GPCR dimers
have been implicated in disease states (Dalrymple et al. 2008). In this study we were
unable to identify any change in constitutive ERK1/2 or AKT signalling, or altered
apoptosis, in PC-3 cells overexpressing combinations of GHS-R1a, GHS-R1b or
GPR39.
In order to assess any protective effect of receptor expression, apoptosis was induced
using tunicamycin, an ER stress inducer, or U0126, an ERK1/2 inhibitor. Treatment
with tunicamycin induced significant apoptosis in the PC-3 cells tested compared to
untreated cells, however, U0126 treatment did not. PC-3 cells have previously been
described to have low levels of basal ERK1/2 phosphorylation, particularly when
compared to other prostate cancer cell lines (Guo et al. 2000; Shimada et al. 2002;
Stangelberger et al. 2005; Ruscica et al. 2006). As there are low levels of ERK1/2
phosphorylation in PC-3 cells, this signalling may not have a significant pro-survival
effect and, therefore, inhibition of this pathway may not lead to significant apoptosis,
as reflected in this study.
Ghrelin, the endogenous ligand of GHS-R1a, has been shown to play a role in cell
survival. Ghrelin had a protective effect when apoptosis was induced in a variety of
ways in a number of different cell types including; doxorubicin- and serum
deprivation-induced apoptosis in cardiomyocytes and endothelial cells (Baldanzi et
al. 2002), serum deprivation-induced apoptosis in adrenal zona glomerulosa cells
164
(Mazzocchi et al. 2004) and adipocytes (Kim et al. 2004), tumor necrosis factor
(TNF)-α-induced apoptosis in mouse osteoblastic MC3T3-E1 cells (Kim et al. 2005)
and vascular smooth muscle cells (Zhang et al. 2008b), doxorubicin-induced
apoptosis in pancreatic β cells (Zhang et al. 2007b), basal apoptosis in the
adrenocortical carcinoma cell line SW-13 (Delhanty et al. 2007), serum deprivation-
and interferon-γ/TNF-α-induced apoptosis in pancreatic β-cells and human
pancreatic islets (Granata et al. 2007), oxygen-glucose deprivation-induced apoptosis
in hypothalamic neuronal cells (Chung et al. 2007) and oxidative stress-induced
apoptosis in cardiomyocytes from adult rats (Liu et al. 2009). In some of these cases
this protective effect was determined to be mediated by the ERK1/2 (Mazzocchi et
al. 2004), AKT (Liu et al. 2009) or by both the ERK1/2 and AKT signalling
pathways (Baldanzi et al. 2002; Kim et al. 2004; Chung et al. 2007; Granata et al.
2007; Zhang et al. 2007b). In some cases, a similar protective effect was observed
for unacylated ghrelin (Baldanzi et al. 2002; Delhanty et al. 2007; Granata et al.
2007; Zhang et al. 2008b) and in cardiomyocytes and endothelial cells, ghrelin-
mediated protection against apoptosis was found to be independent of GHS-R1a
(Baldanzi et al. 2002).
Our research group has previously investigated the role of ghrelin in apoptosis in the
PC-3 prostate cancer cells. Ghrelin had no protective effect on apoptosis induced by
actinomycin D (Yeh et al. 2005). In this study we investigated the potential ghrelin-
independent, constitutive role of the receptor, GHS-R1a, in regulating apoptosis and
whether this effect is modulated by receptor heterodimerisation. In COS-7 or
HEK293 cells transiently transfected with GHS-R1a, a high degree (~50% of
maximal activity) of ligand independent inositol phosphate turnover (Gαq signalling
through the phospholipase C pathway) and activation of cAMP-responsive element
(CRE) gene transcription was observed (Holst et al. 2003) indicating a high degree
of GHS-R1a constitutive signalling. Additional studies by the same group also
determined that GHS-R1a displayed a degree of constitutive signalling through the
serum response element (SRE) pathway and that the receptor is constitutively
internalised in the absence of ligand (Holst et al. 2004). Interestingly, constitutive
phosphorylation of ERK1/2 was not observed in COS-7 cells transiently transfected
with GHS-R1a, however, a clear increase in ERK1/2 phosphorylation was seen after
treatment with ghrelin (Holst et al. 2004). The constitutive signalling of GHS-R1a
165
may play a role in cell survival (Lau et al. 2009). In HEK293 cells stably
overexpressing seabream GHS-R1a, the expression of GHS-R1a significantly
attenuated cadmium induced apoptosis and this protective effect was not modulated
by GHS-R1a ligands (Lau et al. 2009). The protective role of constitutive GHS-R1a
activity was mediated via a protein kinase C-dependent pathway (Lau et al. 2009).
Interestingly, co-expression of GHS-R1b did not alter the survival responses in GHS-
R1a expressing cells (Lau et al. 2009).
Zinc is the only proven ligand for GPR39. The modulation of apoptosis by zinc has
been implicated in the proliferation of malignant cells in prostate cancer. In prostate
cells, zinc has been shown to induce apoptosis (Liang et al. 1999) by inducing
mitochondrial apoptogenesis (Feng et al. 2000). This is interesting, as normal
prostate accumulates the highest amount of zinc of any soft tissue, but the level of
zinc consistently decreases with prostate malignancy (Costello et al. 2005).
Therefore, this decrease in zinc in malignant cells will result in loss of zinc-induced
apoptosis, thereby aiding in the proliferation of malignant cells (Costello et al. 2005).
In this study, we observed an increase in apoptosis when transfected PC-3 cells were
treated with zinc, however, these changes were not statistically significant and did
not appear to be altered by the expression of GPR39 alone, or in combination with
GHS-R1a.
Like GHS-R1a, GPR39 has a high degree of constitutive activity. GPR39 displays
constitutive inositol phosphate turnover (Gαq signalling through the phospholipase C
pathway) and activation of cAMP-responsive element (CRE) gene transcription,
however, the degree of constitutive signalling is lower than that of GHS-R1a (Holst
et al. 2004). GPR39, however, has a higher level of constitutive signalling through
the serum response element (SRE) pathway compared with GHS-R1a (Holst et al.
2004). Like GHS-R1a, constitutive phosphorylation of ERK1/2 was not observed in
COS-7 cells transiently transfected with GPR39, however, an increase in ERK1/2
phosphorylation could be seen after treatment with zinc (Holst et al. 2004). These
findings are in agreement with this study in PC-3 cells where we did not observe
constitutive phosphorylation of ERK1/2 in cells overexpressing GHS-R1a and
GPR39. Interestingly, in contrast to GHS-R1a, GPR39 is not constitutively
internalised and in the absence of agonist it remains at the cell surface (Holst et al.
166
2004). This difference in constitutive internalisation between GHS-R1a and GPR39
was determined to be due to differences in the C-terminal tails (Holliday et al. 2007).
GPR39 has been reported to have a role in the regulation of apoptosis due to this
constitutive activity (Dittmer et al. 2008).
Despite the fact that GHS-R1a and GPR39 are constitutively active and provide a
cell survival effect in some cell types, this was not observed in PC-3 cells in this
study where these receptors and GHS-R1b were overexpressed alone or in
combination. No effect was seen on the basal rate of apoptosis, or on the rate of
apoptosis induced by treatment with tunicamycin. This may indicate that these
receptors do not play a role in apoptosis in prostate cancer cells, at least when
induced by tunicamycin. These receptors could, however, inhibit apoptosis induced
by other methods and may act through signalling pathways other than ERK1/2 and
AKT. Preparation of prostate cancer cell lines stably overexpressing GHS-R1a,
GHS-R1b and GPR39 alone, or in combination, may provide a better experimental
model to investigate these potential effects.
This study focused on the co-expression of the ghrelin receptor isoforms, GHS-R1a
and GHS-R1b and the related receptor, GPR39, and any potential functional
outcomes in prostate cancer cells. Dimers involving GHS-R1a or GHS-R1b have
been shown to attenuate ligand-induced signalling (Chan and Cheng 2004),
constitutive GHS-R1a activity (Chu et al. 2007; Leung et al. 2007; Chow et al. 2008)
and GHS-R1a cell surface expression (Leung et al. 2007; Chow et al. 2008), and to
amplify the signalling of unrelated GPCRs, as is the case for the GHS-R1a/dopamine
receptor dimer (Jiang et al. 2006) and the GHS-R1b/neurotensin receptor 1 dimer,
which functions as a novel receptor type that can signal in response to unrelated
ligands (Takahashi et al. 2006). As GHS-R1a and GPR39 demonstrate high levels of
constitutive activity, the effects of this activity on ERK1/2 and AKT signalling and
apoptosis were investigated in the PC-3 prostate cancer cell line. Overexpression of
GHS-R1a, GHS-R1b and GPR39, alone or in combination, did not increase
constitutive signalling through the ERK1/2 or AKT pathways. In addition,
overexpression of these receptors in PC-3 cells did not significantly alter basal
apoptosis or tunicamycin-induced apoptosis. These results may indeed indicate that
these receptors may not play a role in cell survival in the prostate, when expressed
167
alone or in combination. These receptors could yet have other functions in prostate
cancer, however, the function of GPR39 itself still requires further investigation. The
potential role of GPR39 in the prostate is interesting given that GPR39 is expressed
in this tissue and zinc, a ligand of GPR39, is important in normal prostate biology
and in prostate cancer progression. Rather than targeting the known functions of
GPR39, it may be useful to perform a broader study into GPR39 function in the
prostate, using microarray or proteomic techniques.
168
CHAPTER 7
GENERAL DISCUSSION
169
When this project commenced, the hypothesis that G protein coupled receptors
formed and functioned as homo- and hetero- dimeric units was gaining growing
support in the literature (Hansen and Sheikh 2004; Milligan 2004). This challenged
the dogma that GPCRs generally acted as monomers. It is now believed that
interactions between distinct GPCRs can lead to the formation of novel
pharmacological receptors and can diversify the function of GPCRs (Park and
Palczewski 2005), and a number of novel GPCR dimers have been implicated in the
development of pathophysiological conditions (Dalrymple et al. 2008). Importantly,
novel GPCR dimers represent potential new targets for the development of more
specific, targeted therapeutics for a wide range of diseases.
In this study we investigated the potential for interactions between the ghrelin
receptor, GHS-R1a, with a truncated ghrelin receptor isoform, GHS-R1b, and the
closely related zinc receptor, GPR39, and the potential for functional outcomes in
prostate cancer. Ghrelin and zinc have roles in prostate cancer. Our research group
has shown that ghrelin stimulates proliferation of the PC-3 and LNCaP prostate
cancer cell lines at close to physiological levels (Jeffery et al. 2002; Yeh et al. 2005).
Significantly, ghrelin and the truncated ghrelin receptor isoform, GHS-R1b, are more
highly expressed in prostate cancer when compared with normal prostate tissues
(Jeffery et al. 2002; Yeh et al. 2005). Zinc has a unique role in the biology of the
prostate, where it is normally accumulated at high levels, and the level of zinc
accumulation is greatly decreased in prostate malignancy (Costello and Franklin
2006). This altered accumulation of zinc in prostate cancer provides malignant cells
with significant metabolic, growth and metastatic advantages (Costello and Franklin
2006).
Numerous studies have shown that some GPCR splice variants or C-terminally
truncated mutant GPCRs interact with their corresponding full length wild-type
receptor, (as reviewed in Dalrymple et al. 2008). Additionally, closely related
GPCRs are more likely to form functional heterodimers than less closely related
receptors (Ramsay et al. 2002). We hypothesised, therefore, that as GHS-R1a, GHS-
R1b and GPR39 are very closely related that they will interact. As their ligands are
significant in prostate cancer, we hypothesised that the formation of GHS-R1a/GHS-
R1b and GHS-R1a/GPR39 heterodimers would have significant functional outcomes
170
in prostate cancer development or progression. These novel dimers could, therefore,
provide new targets for the development of potential adjunctive therapeutic
approaches for prostate cancer.
Using a number of experimental techniques we were unable to definitively
demonstrate that GHS-R1a interacts with GHS-R1b or GPR39. Interestingly, the
difficulty in obtaining reliable data with the appropriately controlled methods in this
study reflects the growing controversy regarding the use of these methods which has
emerged in the literature. The formation of GPCR dimers has been largely described
in artificial experimental systems, and this appears to have lead to the over-
interpretation of data in some cases and the over reliance on methodologies that have
significant potential shortcomings (Panetta and Greenwood 2008).
The initial aim of this study was to demonstrate interactions between GHS-R1a,
GHS-R1b and GPR39 using classical co-immunoprecipitation experiments with
differentially tagged receptors (Chapter 3). Using cells co-overexpressing FLAG-
and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we co-immunoprecipitated these
receptors. Although this could be indicative of dimerisation, we interpreted these
data with caution, as we also demonstrated the ability of these receptors to aggregate
during cell lysis and protein solubilisation. This is a commonly reported concern
regarding the co-immunoprecipitation of highly hydrophobic membrane proteins,
and this aggregation could be interpreted as receptor dimerisation (Bouvier 2001;
Devi 2001; Kroeger et al. 2004; Kent et al. 2007; Szidonya et al. 2008). It has been
suggested, therefore, that due to this experimental limitation, additional experimental
techniques should be used to verify receptor-receptor interactions (Szidonya et al.
2008).
At the commencement of this study, the BRET2 system, which offers greater
resolution of the donor and acceptor emission spectrum than first generation BRET,
was described as one of the best available methods to demonstrate GPCR
dimerisation (Mercier et al. 2002; Ramsay et al. 2002), and, therefore, we used this
technique to investigate the potential for GHS-R1a, GHS-R1b and GPR39 to
dimerise (Chapter 4). However, during our studies we encountered a number of
technical limitations. The substrate used during BRET2 studies, coelenterazine 400a,
171
was found to have a low quantum yield and rapid signal decay (Hamdan et al. 2005).
In our study, we observed this phenomenon, where the luminescent and fluorescent
signals rapidly approached the baseline of detection, significantly increasing the
experimental error. While the low quantum yield and rapid signal decay does not
prohibit the use of BRET2 per se, the properties of coelenterazine 400a mean that
high levels of protein expression are required so that a BRET2 signal can be detected
(Kocan et al. 2008). This has significant implications for the physiological relevance
of BRET2 results. The supraphysiological overexpression of donor and acceptor
tagged receptors can lead to ‘bystander BRET’, which is non-specific BRET
resulting from usually non-interacting proteins that are forced into close proximity
due to increased concentrations. To differentiate between specific receptor-receptor
interactions and non-specific interactions a number of BRET controls are required
(James et al. 2006; Pfleger et al. 2006b; Marullo and Bouvier 2007). The results of
these controls which were performed in this study, suggested that the levels of
BRET2 that we observed may in fact be a result of ‘bystander’ BRET. It would be
preferable, therefore, to measure BRET2 in cells expressing lower levels of donor
and acceptor tagged receptor, however, due to the limitations imposed by requiring
the coelenterazine 400a substrate, this would not produce a measurable luminescent
and fluorescent signal. Interestingly, the company that originally supplied and
promoted the BRET2 vectors and substrate (Perkin Elmer) removed it from the
market at the end of 2007 (personal communication, Perkin Elmer). Given the results
of this study, and the reported limitations of the BRET2 methodology, we would
suggest that GPCR dimerisation observed using this method alone may require
further validation.
We investigated the potential for GHS-R1a, GHS-R1b and GPR39 to dimerise using
two different FRET methodologies; acceptor photobleaching FRET (abFRET) and
sensitised emission FRET, (which is the measurement of the acceptor fluorescence
after specific excitation of the donor), by flow cytometry (Chapter 5). Using these
methods we were unable to observe any significant FRET or FRET values that were
likely to result from specific receptor dimerisation. Interestingly, for all receptor
combinations tested, we observed a degree of FRET when measured by acceptor
photobleaching confocal microscopy. The level of FRET observed was within the
reported range observed for almost any pair of integral membrane proteins labelled
172
with a donor and acceptor undergoing random interactions (Vogel et al. 2006).
Our studies were unable to directly and conclusively demonstrate interactions
between GHS-R1a, GHS-R1b and GPR39 (Chapters 3-5). During the course of these
studies, significant concerns regarding the methodologies used to demonstrate GPCR
dimerisation were also raised in the literature, suggesting that more extensive
analysis of these potential interactions was required. Indeed, initial studies using co-
immunoprecipitation and BRET2 could have been interpreted as supporting our
dimerisation hypothesis if these rigorous controls had not been performed. It is
becoming increasingly apparent that a number of the current experimental methods
used to demonstrate GPCR dimerisation are open to interpretation (Gurevich and
Gurevich 2008a) and the unambiguous interpretation of inherently ambiguous data
is currently a major concern in the study of GPCR dimerisation (Gurevich and
Gurevich 2008a). Particularly given the current knowledge of these methodologies, it
is apparent that an extensive understanding of the experimental techniques and also
their potential limitations is required.
Our attempts to directly demonstrate dimerisation between GHS-R1a, GHS-R1b and
GPR39, while inconclusive, do not exclude the possibility that these receptors do
interact, or indeed dimerise. We aimed to further investigate this potential by
demonstrating a functional outcome in prostate cancers cells co-expressing
combinations of these receptors (Chapter 6). The demonstration of specific
functional outcomes from receptor dimerisation is an important step in determining
the physiological significance of receptor interactions. The ERK1/2 and AKT
signalling pathways have been shown to be key pathways in ghrelin mediated cell
survival (Baldanzi et al. 2002; Kim et al. 2004; Mazzocchi et al. 2004; Chung et al.
2007; Granata et al. 2007; Zhang et al. 2007b; Liu et al. 2009) and constitutively
active GHS-R1a and GPR39 can attenuate apoptosis when overexpressed in some
cell types (Dittmer et al. 2008; Lau et al. 2009). We, therefore, investigated the
potential role for GHS-R1a and GPR39 mediated ERK1/2 and AKT constitutive
signalling and apoptosis regulation, and how this may be modulated by receptor
dimerisation in the PC-3 prostate cancer cell line. Overexpression of GHS-R1a,
GHS-R1b and GPR39, alone or in combination, did not increase constitutive
signalling through the ERK1/2 or AKT pathways. In addition, overexpression of
173
these receptors in PC-3 cells did not significantly alter basal apoptosis or
tunicamycin-induced apoptosis. These findings do not support our hypotheses that
GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers have functional outcomes
that may be significant in the development of prostate cancer, although a limited
number of potential functional outcomes were investigated.
As this project progressed, the initial excitement regarding the discovery and early
investigation of GPCR dimerisation has been overshadowed by growing controversy
surrounding this concept. The demonstration that GPCR dimers are involved in the
genesis of disease, gave weight to the argument that GPCR dimerisation is
functionally significant. One of the first physiologically relevant examples was the
demonstration that the type I angiotensin-II receptor (AT1R) and the bradykinin-2
receptor (B2R) heterodimerised resulting in increased AT1R signalling in pre-
eclamptic, hypertensive women (AbdAlla et al. 2001). This was the first disorder
shown to be associated with altered GPCR heterodimerisation, and was published in
Nature Medicine (AbdAlla et al. 2001). This study has often been cited as important
evidence for the significance of GPCR heterodimerisation. Additional studies by the
same group also suggested that this dimer contributes to the angiotensin II hyper-
responsiveness of mesangial cells in experimental hypertension (AbdAlla et al.
2005). Recently, however, researchers from four independent research groups
attempted to reproduce these findings with a view to studying this important
interaction further (Hansen et al. 2009). Using a number of different experimental
methods in a variety of cell types, they found a lack of evidence for AT1R/B2R
heterodimerisation (Hansen et al. 2009). Specifically, they failed to demonstrate any
physical interaction between these receptors, or any functional modulation of AT1R
signalling by B2R in any of the systems tested (Hansen et al. 2009). There is a
striking contrast between the conclusions reached in these recent studies and the
original report and the differences in data have proven difficult to reconcile (Hansen
et al. 2009). This example highlights the need for caution in interpreting data and for
the independent verification of some of the more exciting and significant cases
describing GPCR dimerisation. This is of particular concern for those cases that have
provided fundamental support for the concept of the physiological significance of
GPCR dimerisation.
174
Recent studies have addressed whether or not GPCR dimerisation is required for G
protein activation. These elegant studies were performed by directly incorporating
either one or two GPCRs into a reconstituted phospholipid bilayer and examining G
protein activation (Bayburt et al. 2007; Whorton et al. 2007; Whorton et al. 2008). It
was found that monomeric rhodopsin (Bayburt et al. 2007; Whorton et al. 2008) and
β2-adrenergic receptor (Whorton et al. 2007) were the minimal functional units
required for efficient G protein activation. While these studies do not refute the
theory that GPCR dimers exist, it does suggest that dimerisation is not a requirement
for GPCR signalling and that dimerisation may only play a minor role in G protein
activation (Whorton et al. 2007).
While this study raises significant questions regarding some aspects of GPCR
dimerisation, suggesting that data gained in artificial systems must be interpreted
carefully, it has not been suggested that GPCR dimerisation does not occur. There is
a wealth of experimental evidence to support the concept of GPCR dimerisation
(Milligan 2009). It is becoming clear, however, that greater emphasis needs to be
placed on the demonstration of GPCR dimers in native cellular contexts rather than
in artificial systems that are open to interpretation. Recently the International Union
of Pharmacology Committee on Receptor Nomenclature and Drug Classification
(NU-IUPHAR) outlined a number of criteria that need to be met before a receptor
heterodimer can be accepted by the scientific community (Pin et al. 2007). They
suggest that at least two of the following three criteria need to be demonstrated: 1)
evidence for physical association in native tissue or primary cells; 2) a specific
functional property for the heterodimeric receptor and 3) the use of knockout animals
or RNAi technology demonstrating a significantly altered dimer-mediated response
in the absence of either subunit (Pin et al. 2007). As we were unable to fulfil the
criteria outlined by NC-IUPHAR when considering the findings of this study, we are
unable to conclude that GHS-R1a, GHS-R1b and GPR39 form physiologically
relevant dimers. Interestingly, although a large number of GPCR dimers have been
described in the literature, the only examples to currently meet all of these criteria are
the obligate heterodimer class C GPCRs, the GABAB receptor
(GABABR1/GABABR2), the sweet taste receptor (T1R2/T1R3) and the umami taste
receptor (T1R1/T1R3) (Milligan 2009). Unambiguous evidence regarding the largest
class of GPCRs, the class A receptors to which the ghrelin receptor family belongs,
175
is sparse (Gurevich and Gurevich 2008b).
A number of avenues are available to investigate further the ability of GHS-R1a,
GHS-R1b and GPR39 to form functionally relevant dimers. In this study we used the
BRET2 methodology, however, it is now apparent that there are major limitations to
this technique. During the course of this study, improvements to BRET2 have been
suggested, where a novel form of luciferase, Rluc2 or Rluc8, is used as the energy
donor, giving greater signal intensity and stability following the addition of the
coelenterazine 400a substrate (Kocan et al. 2008). Other BRET methods that may
represent significant improvements are also now available, including extended
bioluminescence resonance energy transfer (eBRET) (Pfleger et al. 2006a) and
BRET3 (De et al. 2009). Additionally, as RET is based not only on the proximity of
the donor and the acceptor, but also on their relative orientations, redesigning our
current BRET2 and FRET constructs with a range of combinations of different linker
sequences between the receptor and the tagged fluorophore, may also result in a
different RET signal (Szidonya et al. 2008). It is unclear, however, if these
approaches would lead to the identification of dimerisation and this may not be
functionally significant. Perhaps a more reasonable approach would be to investigate
further potential functional outcomes of GHS-R1a/GHS-R1b and GHS-R1a/GPR39
heterodimersation in cells co-overexpressing these receptors using a broader
microarray or proteomic approach, as it is difficult to predict the functional outcome
of dimerisation. These findings would need to be confirmed in a native context,
potentially using RNAi methods for targeted knockdown of the relevant receptor.
However, given the current controversy regarding the propensity of class A GPCRs
to form and function as heterodimers, additional studies may not be warranted.
It is possible that the action of both ghrelin and zinc may be independent of GHS-
R1a and GPR39 in the prostate. There is increasing evidence that, in addition to
GHS-R1a, there is an alternative receptor which binds both ghrelin and its des-acyl
form (Cassoni et al. 2001; Baldanzi et al. 2002; Bedendi et al. 2003; Cassoni et al.
2004; Cassoni et al. 2006; Delhanty et al. 2006; Martini et al. 2006; Sato et al. 2006;
Filigheddu et al. 2007; Granata et al. 2007). Ghrelin actions in the prostate could be
mediated by the putative alternative ghrelin receptor. Additionally, in the preliminary
studies presented in this thesis, we were unable to demonstrate a GPR39-mediated,
176
zinc function in prostate cancer (Chapter 6). We cannot rule out, therefore, that the
negative findings of this study of the functional outcomes of GHS-R1a and GPR39
expression in the prostate may simply reflect that these receptors do not play a role in
the ghrelin and zinc mediated functions in the prostate.
There is currently an urgent need for better prognostic and diagnostic markers and
better adjuvant therapies for prostate cancer. The role of ghrelin and zinc in the
prostate remains interesting and requires further investigation. The increased
expression of ghrelin in prostate cancer provides a potential therapeutic target (Yeh
et al. 2005; Lanfranco et al. 2008). The targeted inhibition of ghrelin, potentially by
inhibition of the newly discovered ghrelin-acylating enzyme, ghrelin O-acyl
transferase (GOAT), may provide a mechanism for modulating prostate cancer
growth. GOAT expression has been demonstrated in the prostate (personal
communication, Dr. Inge Siem). Zinc may also provide a novel biomarker for the
screening of prostate cancer (Costello and Franklin 2009). Interestingly, a europium
luminescence assay that can accurately determine the levels of citrate in microlitre
volumes of prostate fluid has recently been developed (Pal et al. 2009). The levels of
citrate in the prostate are directly linked with the levels of zinc, as zinc increases
citrate production. This test is an exciting development, as it may be used to indicate
the onset or progression of prostate cancer (Pal et al. 2009). Further research into the
role of zinc in prostate cancer may also provide novel directions for the development
of new therapeutic drugs (Costello and Franklin 2006).
In recent years the growing excitement regarding the potential of newly discovered
GPCR dimers has been tempered by concerns regarding the validity of a great deal of
data that has been generated in this field. GPCR dimerisation provides the potential
for an increasing diversity of GPCR functions that may provide avenues for the
development of novel heterodimer-specific drugs. Ultimately, this may provide new
medicines that are more selective and have reduced side effects (Kent et al. 2007).
Importantly, however, it will be necessary for basic researchers to unambiguously
identify these drug targets and definitively demonstrate their relevance in vivo with
significant physiological outcomes (Kent et al. 2007). It has become increasingly
clear, therefore, that the methods applied must yield conclusive answers and the
temptation to over-interpret experimental data must be avoided (Gurevich and
177
Gurevich 2008a). Currently, there is significant controversy regarding the ability of
class A GPCRs to form functional dimers. While some scientists now believe that
GPCR dimerisation may not occur at all, at the other extreme, some authors still
hypothesise that GPCRs may always function as dimers (Chabre and le Maires 2005;
Fotiadis et al. 2006; Gurevich and Gurevich 2008b). It is most likely that while
GPCR dimers may form in some specific cases, they are unlikely to occur as
ubiquitously as once imagined. It is becoming increasingly unlikely that a general
model to describe GPCR dimerisation and its mechanisms will be described and it is
more likely that individual GPCR dimers will need to be assessed on a case by case
basis. However, given the potential for alternative functional outcomes, the ability of
GPCRs to dimerise is likely to remain a focus for intense research for a number of
years to come. As our study has not demonstrated specific interactions between
GHS-R1a, GHS-R1b and GPR39, it is possible that these receptors do not form
physiologically significant dimers. In contrast to our study, human GHS-R1a and
GHS-R1b were recently directly demonstrated to heterodimerise (Leung et al. 2007).
This discrepancy with our findings could be due to differences in interpretation of the
co-immunoprecipitation and BRET2 data. While we cannot conclusively prove that
GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers do not form, our findings
are supported by recent opinions being expressed in the literature casting doubt on
class A GPCR heterodimerisation. The development of new, more robust
technologies is required to resolve this issue in the future. Importantly, we believe,
that given the current knowledge of the potential limitations of the co-
immunoprecipitation and resonance energy transfer methodologies, cautious
interpretation of such data is required. This would avoid spurious additional research
being performed that may be based on weak fundamental data. Given the important
role of ghrelin and zinc in the progression of prostate cancer, the receptors, GHS-
R1a, GHS-R1b and GPR39, may yet provide novel targets for the development of
adjuvant prostate cancer therapeutics.
178
CHAPTER 8
REFERENCES
179
AbdAlla, S., Abdel-Baset, A., Lother, H., El Massiery, A. and Quitterer, U. (2005). Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension. Journal of Molecular Neuroscience 26(2-3): 185-192.
AbdAlla, S., Lother, H., el Massiery, A. and Quitterer, U. (2001). Increased AT1
receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nature Medicine 7(9): 1003-1009.
AbdAlla, S., Lother, H. and Quitterer, U. (2000). AT1-receptor heterodimers show
enhanced G-protein activation and altered receptor sequestration. Nature 407(6800): 94-98.
Adeghate, E. and Ponery, A. (2002). Ghrelin stimulates insulin secretion from the
pancreas of normal and diabetic rats. Journal of Neuroendocrinology 14(7): 555-560.
Ahn, S., Shenoy, S., Wei, H. and Lefkowitz, R. (2004). Differential Kinetic and
Spatial Patterns of ß-Arrestin and G Protein-mediated ERK Activation by the Angiotensin II Receptor. Journal of Biological Chemistry 279(34): 35518-35525.
Andersson, U., Filipsson, K., Abbott, C., Woods, A., Smith, K., Bloom, S., Carling,
D. and Small, C. (2004). AMP-activated protein kinase plays a role in the control of food intake. Journal of Biological Chemistry 279(13): 12005-12008.
Andreis, P., Malendowicz, L., Trejter, M., Neri, G., Spinazzi, R., Rossi, G. and
Nussdorfer, G. (2003). Ghrelin and growth hormone secretagogue receptor are expressed in the rat adrenal cortex: evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells. FEBS letters 536(1-3): 173-179.
Angers, S., Salahpour, A. and Bouvier, M. (2002). Dimerization: An emerging
concept for G protein-coupled receptor ontogeny and function. Annual Reviews in Pharmacology and Toxicology 42(1): 409-435.
Asakawa, A., Inui, A., Fujimiya, M., Sakamaki, R., Shinfuku, N., Ueta, Y., Meguid,
M. and Kasuga, M. (2005). Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin. Gut 54(1): 18-24.
Asakawa, A., Inui, A., Kaga, T., Katsuura, G., Fujimiya, M., Fujino, M. and Kasuga,
M. (2003). Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 52(7): 947-952.
Asakawa, A., Inui, A., Kaga, T., Yuzuriha, H., Nagata, T., Fujimiya, M., Katsuura,
G., Makino, S., Fujino, M. and Kasuga, M. (2001a). A role of ghrelin in neuroendocrine and behavioral responses to stress in mice. Neuroendocrinology 74: 143-147.
180
Asakawa, A., Inui, A., Kaga, T., Yuzuriha, H., Nagata, T., Ueno, N., Makino, S., Fujimiya, M., Niijima, A. and Fujino, M. (2001b). Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120(2): 337-345.
Ataka, K., Inui, A., Asakawa, A., Kato, I. and Fujimiya, M. (2008). Obestatin
inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. American Journal of Physiology- Gastrointestinal and Liver Physiology 294(5): G1210-1218.
Babcock, G., Farzan, M. and Sodroski, J. (2003). Ligand-independent dimerization
of CXCR4, a principal HIV-1 coreceptor. Journal of Biological Chemistry 278(5): 3378-3385.
Bacart, J., Corbel, C., Jockers, R., Bach, S. and Couturier, C. (2008). The BRET
technology and its application to screening assays. Biotechnology Journal 3(3): 311 - 324.
Baiguera, S., Conconi, M., Guidolin, D., Mazzocchi, G., Malendowicz, L.,
Parnigotto, P., Spinazzi, R. and Nussdorfer, G. (2004). Ghrelin inhibits in vitro angiogenic activity of rat brain microvascular endothelial cells. International Journal of Molecular Medicine 14(5): 849-854.
Malan, D., Baj, G., Granata, R. and Broglio, F. (2002). Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. Journal of Cell Biology 159(6): 1029-1037.
Bang, A., Soule, S., Yandle, T., Richards, A. and Pemberton, C. (2006).
Characterisation of proghrelin peptides in mammalian tissue and plasma. Journal of Endocrinology 192(2): 313-323.
Barki-Harrington, L., Bookout, A., Wang, G., Lamb, M., Leeb-Lundberg, L. and
Daaka, Y. (2003). Requirement for direct cross-talk between B1 and B2 kinin receptors for the proliferation of androgen-insensitive prostate cancer PC3 cells. Biochemical Journal 371(2): 581-587.
Barreiro, M., Gaytan, F., Castellano, J., Suominen, J., Roa, J., Gaytan, M., Aguilar,
E., Dieguez, C., Toppari, J. and Tena-Sempere, M. (2004). Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 145(11): 4825-4834.
Bassil, A., Häglund, Y., Brown, J., Rudholm, T., Hellström, P., Näslund, E., Lee, K.
and Sanger, G. (2006). Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract. British Journal of Pharmacology 150(1): 58-64.
Bastiaens, P., Majoul, I., Verveer, P., Söling, H. and Jovin, T. (1996). Imaging the
intracellular trafficking and state of the AB5 quaternary structure of cholera
181
toxin. The EMBO Journal 15(16): 4246-4253. Bayburt, T., Leitz, A., Xie, G., Oprian, D. and Sligar, S. (2007). Transducin
activation by nanoscale lipid bilayers containing one and two rhodopsins. Journal of Biological Chemistry 282(20): 14875-14881.
Bedendi, I., Alloatti, G., Marcantoni, A., Malan, D., Catapano, F., Ghé, C.,
Deghenghi, R., Ghigo, E. and Muccioli, G. (2003). Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin. European Journal of Pharmacology 476(1-2): 87-95.
Berglund, M., Schober, D., Esterman, M. and Gehlert, D. (2003). Neuropeptide Y Y4
receptor homodimers dissociate upon agonist stimulation. Journal of Pharmacology and Experimental Therapeutics 307(3): 1120-1126.
Bertaccini, A., Pernetti, R., Marchiori, D., Pagotto, U., Palladoro, F., Palmieri, F.,
Vitullo, G., Guidi, M. and Martorana, G. (2009). Variations in blood ghrelin levels in prostate cancer patients submitted to hormone suppressive treatment. Anticancer Research 29(4): 1345-1348.
Bertrand, G. and Vladesco, R. (1921). Intervention probable du zinc dans les
phenomenes de fecondation chez les animaux vertebres. Comptes Rendus de l'Académie des Sciences 173: 176–179.
Bertrand, L., Parent, S., Caron, M., Legault, M., Joly, E., Angers, S., Bouvier, M.,
Brown, M., Houle, B. and Ménard, L. (2002). The BRET2/arrestin assay in stable recombinant cells: A platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). Journal of Receptors and Signal Transduction 22(1): 533-541.
Black, P. C., Mize, G. J., Karlin, P., Greenberg, D. L., Hawley, S. J., True, L. D.,
Vessella, R. L. and Takayama, T. K. (2007). Overexpression of protease-activated receptors-1,-2, and-4 (PAR-1, -2, and -4) in prostate cancer. Prostate 67(7): 743-56.
Blackburn, P., Simpson, C., Nibbs, R., O'Hara, M., Booth, R., Poulos, J., Isaacs, N.
and Graham, G. (2004). Purification and biochemical characterization of the D6 chemokine receptor. Biochemical Journal 379(2): 263-272.
Blank, N., Burger, R., Duerr, B., Bakker, F., Wohlfarth, A., Dumitriu, I., Kalden, J.
R. and Herrmann, M. (2002). MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death. Cytometry 48(4): 179-84.
Borjigin, J. and Nathans, J. (1994). Insertional mutagenesis as a probe of rhodopsin's
topography, stability, and activity. Journal of Biological Chemistry 269(20): 14715-14722.
Bouvier, M., Heveker, N., Jockers, R., Marullo, S. and Milligan, G. (2007). BRET
analysis of GPCR oligomerization: newer does not mean better. Nature
182
Methods 4(1): 3. Bouvier, M. (2001). Oligomerization of G-protein-coupled transmitter receptors.
Nature Reviews Neuroscience 2(4): 274-286. Bowers, C., Momany, F., Reynolds, G. and Hong, A. (1984). On the in vitro and in
vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114(5): 1537-1545.
Breit, A., Lagace, M. and Bouvier, M. (2004). Hetero-oligomerization between ß2-
and ß3-adrenergic receptors generates a ß-adrenergic signalling unit with distinct functional properties. Journal of Biological Chemistry 279(27): 28756-28765.
Brescianu, E., Rapetti, D., Dona, F., Bulgarelli, I., Tamiazzo, L., Locatelli, V. and
Torsello, A. (2006). Obestatin inhibits feeding but does not modulate GH and corticosterone secretion in the rat. Journal of Endocrinological Investigation 29(8): RC16-18.
Bridges, T. M. and Lindsley, C. W. (2008). G-protein-coupled receptors: from
classical modes of modulation to allosteric mechanisms. ACS Chemical Biology 3(9): 530-41.
Broglio, F., Arvat, E., Benso, A., Gottero, C., Muccioli, G., Papotti, M., Lely, A.,
Deghenghi, R. and Ghigo, E. (2001). Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. Journal of Clinical Endocrinology & Metabolism 86(10): 5083-5083.
Bullis, B., Li, X., Rieder, C., Singh, D., Berthiaume, L. and Fliegel, L. (2002).
Properties of the Na+/H+ exchanger protein detergent-resistant aggregation and membrane microdistribution. FEBS Letters 269(19): 4887-4895.
Camina, J., Campos, J., Caminos, J., Dieguez, C. and Casanueva, F. (2007a).
Obestatin-mediated proliferation of human retinal pigment epithelial cells: regulatory mechanisms. Journal of Cellular Physiology 211(1): 1-9.
Camina, J., Lodeiro, M., Ischenko, O., Martini, A. and Casanueva, F. (2007b).
Stimulation by ghrelin of p42/p44 mitogen-activated protein kinase through the GHS-R1a receptor: Role of G-proteins and ß-arrestins. Journal of Cellular Physiology 213(1): 187-200.
Camina, J. (2006). Cell biology of the ghrelin receptor. Journal of
Neuroendocrinology 18(1): 65-76. Canals, M., Lopez-Gimenez, J. and Milligan, G. (2009). Cell surface delivery and
structural re-organization by pharmacological chaperones of an oligomerization-defective α1b-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers. Biochemical Journal 417(1): 161-172.
183
Carlini, V., Schiöth, H. and debarioglio, S. (2007). Obestatin improves memory performance and causes anxiolytic effects in rats. Biochemical and Biophysical Research Communications 352(4): 907-912.
Carlini, V., Monzón, M., Varas, M., Cragnolini, A., Schiöth, H., Scimonelli, T. and
de Barioglio, S. (2002). Ghrelin increases anxiety-like behavior and memory retention in rats. Biochemical and Biophysical Research Communications 299(5): 739-743.
Cassoni, P., Allia, E., Marrocco, T., Ghe, C., Ghigo, E., Muccioli, G. and Papotti, M.
(2006). Ghrelin and cortistatin in lung cancer: expression of peptides and related receptors in human primary tumors and in vitro effect on the H345 small cell carcinoma cell line. Journal of Endocrinology Investigation 29(9): 781-90.
Cassoni, P., Ghe, C., Marrocco, T., Tarabra, E., Allia, E., Catapano, F., Deghenghi,
R., Ghigo, E., Papotti, M. and Muccioli, G. (2004). Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. European Journal of Endocrinology 150(2): 173-184.
Cassoni, P., Papotti, M., Ghe, C., Catapano, F., Sapino, A., Graziani, A., Deghenghi,
R., Reissmann, T., Ghigo, E. and Muccioli, G. (2001). Identification, characterization, and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. Journal of Clinical Endocrinology & Metabolism 86(4): 1738-1745.
Chabre, M., Deterre, P. and Antonny, B. (2009). The apparent cooperativity of some
GPCRs does not necessarily imply dimerization. Trends in Pharmacological Sciences 30(4): 182-187.
Chabre, M. and le Maires, M. (2005). Monomeric G-protein-coupled receptor as a
functional unit. Biochemistry 44(27): 9395-9403. Chabre, M., Cone, R. and Saibil, H. (2003). Biophysics (communication arising): Is
rhodopsin dimeric in native retinal rods? Nature 426(6962): 30-31. Chan, C. and Cheng, C. (2004). Identification and functional characterization of two
alternatively spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream Acanthopagrus schlegeli. Molecular and Cellular Endocrinology 214(1-2): 81-95.
Chan, F., Siegel, R., Zacharias, D., Swofford, R., Holmes, K., Tsien, R. and Lenardo,
M. (2001). Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signalling using spectral variants of the green fluorescent protein. Cytometry 44(4): 361-368.
Chang, J. and Korolev, V. (1996). Specific toxicity of tunicamycin in induction of
programmed cell death of sympathetic neurons. Experimental Neurology
184
137(2): 201-211. Chartrel, N., Alvear-Perez, R., Leprince, J., Iturrioz, X., Reaux-Le Goazigo, A.,
Audinot, V., Chomarat, P., Coge, F., Nosjean, O., Rodriguez, M., Galizzi, J. P., Boutin, J. A., Vaudry, H. and Llorens-Cortes, C. (2007). Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake". Science 315(5813): 766; author reply 766.
Chen, C., Chien, E., Chang, F., Lu, C., Luo, J. and Lee, S. (2008). Impacts of
peripheral obestatin on colonic motility and secretion in conscious fed rats. Peptides 29(9): 1603-1608.
Chen, C., Inui, A., Asakawa, A., Fujino, K., Kato, I., Chen, C., Ueno, N. and
Fujimiya, M. (2005). Des-acyl ghrelin acts by CRF type 2 receptors to disrupt fasted stomach motility in conscious rats. Gastroenterology 129(1): 8-25.
Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S.,
Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K. and Stevens, R. C. (2007). High-resolution crystal structure of an engineered human ß2-adrenergic G protein-coupled receptor. Science 318(5854): 1258-65.
Choi, K., Roh, S., Hong, Y., Shrestha, Y., Hishikawa, D., Chen, C., Kojima, M.,
Kangawa, K. and Sasaki, S. (2003). The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis. Endocrinology 144(3): 754-759.
Chow, K., Leung, P., Cheng, C., Cheung, W. and Wise, H. (2008). The constitutive
activity of ghrelin receptors is decreased by co-expression with vasoactive prostanoid receptors when over-expressed in human embryonic kidney 293 cells. International Journal of Biochemistry and Cell Biology 40(11): 2627-2637.
Chu, K., Chow, K., Leung, P., Lau, P., Chan, C., Cheng, C. and Wise, H. (2007).
Over-expression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on. International Journal of Biochemistry and Cell Biology 39(4): 752-764.
Chung, H., Kim, E., Lee, D., Seo, S., Ju, S., Lee, D., Kim, H. and Park, S. (2007).
Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen-glucose deprivation. Endocrinology 148(1): 148-159.
Mancini, G., Lutz, B., Bergh, A. and Fowler, C. (2009). A high cannabinoid CB1 receptor immunoreactivity is associated with disease severity and outcome in prostate cancer. European Journal of Cancer 45(1): 174-182.
Clegg, R., Murchie, A., Zechel, A. and Lilley, D. (1993). Observing the helical
geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proceedings of the National Academy of Sciences 90(7): 2994-2998.
185
Conconi, M., Nico, B., Guidolin, D., Baiguera, S., Spinazzi, R., Rebuffat, P.,
Malendowicz, L., Vacca, A., Carraro, G. and Parnigotto, P. (2004). Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo. Peptides 25(12): 2179-2185.
Costello, L. and Franklin, R. (2009). Prostatic fluid electrolyte composition for the
screening of prostate cancer: a potential solution to a major problem. Prostate Cancer and Prostatic Diseases 12: 17-24.
Costello, L. and Franklin, R. (2006). The clinical relevance of the metabolism of
prostate cancer; zinc and tumor suppression: connecting the dots. Molecular Cancer 5: 17.
Costello, L., Franklin, R., Feng, P., Tan, M. and Bagasra, O. (2005). Zinc and
prostate cancer: A critical scientific, medical, and public interest issue (United States). Cancer Causes and Control 16(8): 901-915.
Costello, L., Liu, Y., Franklin, R. and Kennedy, M. (1997). Zinc inhibition of
mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. Journal of Biological Chemistry 272(46): 28875-28881.
Couve, A., Filippov, A., Connolly, C., Bettler, B., Brown, D. and Moss, S. (1998).
Intracellular retention of recombinant GABAB receptors. Journal of Biological Chemistry 273(41): 26361-26367.
Cowley, M., Smith, R., Diano, S., Tschöp, M., Pronchuk, N., Grove, K., Strasburger,
C., Bidlingmaier, M., Esterman, M. and Heiman, M. (2003). The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37(4): 649-661.
Cummings, D., Purnell, J., Frayo, R., Schmidova, K., Wisse, B. and Weigle, D.
(2001). A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50(8): 1714-1719.
Czifra, G., Varga, A., Nyeste, K., Marincsák, R., Tóth, B., Kovács, I., Kovács, L. and
Bíró, T. (2009). Increased expressions of cannabinoid receptor-1 and transient receptor potential vanilloid-1 in human prostate carcinoma. Journal of Cancer Research and Clinical Oncology 135(4): 507-514.
Daaka, Y. (2004). G proteins in cancer: the prostate cancer paradigm. Science's
Signal Transduction Knowledge Environment 2004(216): re2. Dacres, H., Dumancic, M., Horne, I. and Trowell, S. (2009). Direct comparison of
fluorescence-and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage. Biosensors and Bioelectronics 24(5): 1164-1170.
Dacres, H., Dumancic, M., Horne, I. and Trowell, S. (2008). Direct comparison of
bioluminescence-based resonance energy transfer methods for monitoring of
186
proteolytic cleavage. Analytical Biochemistry 385(2): 194-202. Dalrymple, M., Pfleger, K. and Eidne, K. (2008). G protein-coupled receptor dimers:
Functional consequences, disease states and drug targets. Pharmacology and Therapeutics 118(3): 359-371.
Date, Y., Nakazato, M., Hashiguchi, S., Dezaki, K., Mondal, M. S., Hosoda, H.,
Kojima, M., Kangawa, K., Arima, T., Matsuo, H., Yada, T. and Matsukura, S. (2002). Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes 51(1): 124-9.
Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M., Suganuma, T.,
Matsukura, S., Kangawa, K. and Nakazato, M. (2000). Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141(11): 4255-4261.
Day, R. and Schaufele, F. (2008). Fluorescent protein tools for studying protein
dynamics in living cells: a review. Journal of Biomedical Optics 13(3): 031202.
De, A., Ray, P., Loening, A. and Gambhir, S. (2009). BRET3: a red-shifted
bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals. The FASEB Journal.
De Smet, B., Thijs, T., Peeters, T. and Depoortere, I. (2007). Effect of peripheral
obestatin on gastric emptying and intestinal contractility in rodents. Neurogastroenterology and Motility 19(3): 211-217.
De Vries, L., Zheng, B., Fischer, T., Elenko, E. and Farquhar, M. (2000). The
regulator of G protein signalling family. Annual Review of Pharmacology and Toxicology 40(1): 235-271.
De Vriese, C., Gregoire, F., De Neef, P., Robberecht, P. and Delporte, C. (2005).
Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology 146(3): 1514-1522.
DeBoer, M. (2008). Emergence of ghrelin as a treatment for cachexia syndromes.
Nutrition 24(9): 806-814. Delhanty, P., van Koetsveld, P., Gauna, C., van de Zande, B., Vitale, G., Hofland, L.
and van der Lely, A. (2007). Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway. American Journal of Physiology- Endocrinology And Metabolism 293(1): E302-E309.
Delhanty, P., van der Eerden, B., van der Velde, M., Gauna, C., Pols, H., Jahr, H.,
Chiba, H., Van der Lely, A. and van Leeuwen, J. (2006). Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated
187
protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a. Journal of Endocrinology 188(1): 37-47.
Desouki, M., Geradts, J., Milon, B., Franklin, R. and Costello, L. (2007). hZip 2 and
hZip 3 zinc transporters are down regulated in human prostate adenocarcinomatous glands. Molecular Cancer 6(1): 37.
Devi, L. (2001). Heterodimerization of G-protein-coupled receptors: pharmacology,
signalling and trafficking. Trends in Pharmacological Sciences 22(10): 532-537.
DeWire, S., Ahn, S., Lefkowitz, R. and Shenoy, S. (2007). ß-arrestins and cell
signalling. Dinger, M., Bader, J., Kobor, A., Kretzschmar, A. and Beck-Sickinger, A. (2003).
Homodimerization of neuropeptide Y receptors investigated by fluorescence resonance energy transfer in living cells. Journal of Biological Chemistry 278(12): 10562-10571.
Dittmer, S., Sahin, M., Pantlen, A., Saxena, A., Toutzaris, D., Pina, A., Geerts, A.,
Golz, S. and Methner, A. (2008). The constitutively active orphan G-protein-coupled receptor GPR39 protects from cell death by increasing secretion of pigment epithelium-derived growth factor. Journal of Biological Chemistry 283(11): 7074-7081.
Dixit, V., Schaffer, E., Pyle, R., Collins, G., Sakthivel, S., Palaniappan, R., Lillard, J.
and Taub, D. (2004). Ghrelin inhibits leptin-and activation-induced proinflammatory cytokine expression by human monocytes and T cells. Journal of Clinical Investigation 114(1): 57-66.
Drake, M., Shenoy, S. and Lefkowitz, R. (2006). Trafficking of G protein-coupled
receptors. Circulation Research 99(6): 570-582. Dudek, H., Datta, S., Franke, T., Birnbaum, M., Yao, R., Cooper, G., Segal, R.,
Kaplan, D. and Greenberg, M. (1997). Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275(5300): 661-665.
Duxbury, M., Waseem, T., Ito, H., Robinson, M., Zinner, M., Ashley, S. and Whang,
E. (2003). Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochemical and Biophysical Research Communications 309(2): 464-468.
Dye, B. (2005). Flow cytometric analysis of CFP–YFP FRET as a marker for in vivo
protein–protein interaction. Clinical and Applied Immunology Reviews 5(5): 307-324.
Egerod, K. L., Holst, B., Petersen, P. S., Hansen, J. B., Mulder, J., Hokfelt, T. and
Schwartz, T. W. (2007). GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue. Molecular Endocrinology 21(7):
188
1685-1698. Elbein, A. (1987). Inhibitors of the biosynthesis and processing of N-linked
oligosaccharide chains. Annual Review of Biochemistry 56(1): 497-534. Ellis, J., Pediani, J., Canals, M., Milasta, S. and Milligan, G. (2006). Orexin-1
receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and-independent coordinated alterations of receptor localization and function. Journal of Biological Chemistry 281(50): 38812-38824.
Esler, W. P., Rudolph, J., Claus, T. H., Tang, W., Barucci, N., Brown, S. E., Bullock,
W., Daly, M., Decarr, L., Li, Y., Milardo, L., Molstad, D., Zhu, J., Gardell, S. J., Livingston, J. N. and Sweet, L. J. (2007). Small-molecule ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss. Endocrinology 148(11): 5175-5185.
Fan, T., Varghese, G., Nguyen, T., Tse, R., O'Dowd, B. and George, S. (2005). A
Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of μ and δ Opioid Receptor Hetero-oligomers. Journal of Biological Chemistry 280(46): 38478-38488.
Feng, P., Liang, J., Li, T., Guan, Z., Zou, J., Franklin, R. and Costello, L. (2000).
Filigheddu, N., Gnocchi, V., Coscia, M., Cappelli, M., Porporato, P., Taulli, R.,
Traini, S., Baldanzi, G., Chianale, F. and Cutrupi, S. (2007). Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells. Molecular Biology of the Cell 18(3): 986-994.
Fiorentini, C., Busi, C., Gorruso, E., Gotti, C., Spano, P. and Missale, C. (2008).
Reciprocal regulation of dopamine D1 and D3 receptor function and trafficking by heterodimerization. Molecular Pharmacology 74(1): 59-69.
Fitzpatrick, J., Schulman, C., Zlotta, A. and Schroder, F. (2009). Prostate cancer: a
serious disease suitable for prevention. BJU international 103(7): 864-870. Floyd, D., Geva, A., Bruinsma, S., Overton, M., Blumer, K. and Baranski, T. (2003).
C5a Receptor Oligomerization II - Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast. Journal of Biological Chemistry 278(37): 35354-35361.
Foord, S., Bonner, T., Neubig, R., Rosser, E., Pin, J., Davenport, A., Spedding, M.
and Harmar, A. (2005). International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacological Reviews 57(2): 279-288.
Forster, T. (1948). Zwischenmolekulare energiewanderung und fluoreszenz. Annalen
der Physik 437(1-2): 55-75. Fotiadis, D., Jastrzebska, B., Philippsen, A., Müller, D., Palczewski, K. and Engel,
189
A. (2006). Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors. Current Opinion in Structural Biology 16(2): 252-259.
Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D., Engel, A. and Palczewski, K.
Franklin, R., Feng, P., Milon, B., Desouki, M., Singh, K., Kajdacsy-Balla, A.,
Bagasra, O. and Costello, L. (2005). hZIP 1 zinc uptake transporter down regulation and zinc depletion in prostate cancer. Molecular Cancer 4(1): 32.
Fredriksson, R., Lagerstrom, M., Lundin, L. and Schioth, H. (2003). The G-protein-
coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Molecular Pharmacology 63(6): 1256-1272.
Takeda, S., Takeuchi, Y., Fukumoto, S. and Kangawa, K. (2004). Ghrelin directly regulates bone formation. Journal of Bone and Mineral Research 20(5): 790-798.
Gandiá, J., Lluis, C., Ferre, S., Franco, R. and Ciruela, F. (2008). Light resonance
energy transfer-based methods in the study of G protein-coupled receptor oligomerization. BioEssays 30: 82-89.
Gaskin, F., Farr, S., Banks, W., Kumar, V. and Morley, J. (2003). Ghrelin-induced
feeding is dependent on nitric oxide. Peptides 24(6): 913-918. Gauna, C., Kiewiet, R., Janssen, J., van de Zande, B., Delhanty, P., Ghigo, E.,
Hofland, L., Themmen, A. and van der Lely, A. (2007). Unacylated ghrelin acts as a potent insulin secretagogue in glucose-stimulated conditions. American Journal of Physiology- Endocrinology And Metabolism 293(3): E697.
Gehret, A., Bajaj, A., Naider, F. and Dumont, M. (2006). Oligomerization of the
yeast α-factor receptor: implications for dominant negative effects of mutant receptors. Journal of Biological Chemistry 281(30): 20698-20714.
George, S., O'Dowd, B. and Lee, S. (2002). G-protein-coupled receptor
oligomerization and its potential for drug discovery. Nature Reviews Drug Discovery 1(10): 808-820.
Gloriam, D., Fredriksson, R. and Schiöth, H. (2007). The G protein-coupled receptor
subset of the rat genome. BMC genomics 8(1): 338. Gnanapavan, S., Kola, B., Bustin, S., Morris, D., McGee, P., Fairclough, P.,
Bhattacharya, S., Carpenter, R., Grossman, A. and Korbonits, M. (2002). The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. Journal of Clinical Endocrinology & Metabolism 87(6): 2988-2988.
190
Golovine, K., Makhov, P., Uzzo, R., Shaw, T., Kunkle, D. and Kolenko, V. (2008).
Overexpression of the Zinc Uptake Transporter hZIP1 Inhibits Nuclear Factor-κ B and Reduces the Malignant Potential of Prostate Cancer Cells In vitro and In vivo. Clinical Cancer Research 14(17): 5376-5384.
Gomella, L. G., Johannes, J. and Trabulsi, E. J. (2009). Current prostate cancer
treatments: effect on quality of life. Urology 73(5 Suppl): S28-35. Gomes, I., Jordan, B., Gupta, A., Trapaidze, N., Nagy, V. and Devi, L. (2000).
Heterodimerization of μ and δ opioid receptors: a role in opiate synergy. Journal of Neuroscience 20(22): 110-110.
Zhou, M., Okawa, Y., Callado, L. and Milligan, G. (2008). Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature 452(7183): 93-97.
Gorshkova, E., Erokhina, T., Stroganova, T., Yelina, N., Zamyatnin, A., Kalinina,
N., Schiemann, J., Solovyev, A. and Morozov, S. (2003). Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata. Journal of General Virology 84(4): 985-994.
Gourcerol, G., St-Pierre, D. H. and Tache, Y. (2007). Lack of obestatin effects on
food intake: should obestatin be renamed ghrelin-associated peptide (GAP)? Regulatory Peptides 141(1-3): 1-7.
Gourcerol, G., Million, M., Adelson, D., Wang, Y., Wang, L., Rivier, J., St-Pierre, D.
and Tache, Y. (2006). Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signalling in rodents. Peptides 27(11): 2811-2819.
Granata, R., Settanni, F., Gallo, D., Trovato, L., Biancone, L., Cantaluppi, V., Nano,
R., Annunziata, M., Campiglia, P., Arnoletti, E., Ghe, C., Volante, M., Papotti, M., Muccioli, G. and Ghigo, E. (2008). Obestatin promotes survival of pancreatic beta-cells and human islets and induces expression of genes involved in the regulation of beta-cell mass and function. Diabetes 57(4): 967-979.
Granata, R., Settanni, F., Biancone, L., Trovato, L., Nano, R., Bertuzzi, F.,
Destefanis, S., Annunziata, M., Martinetti, M., Catapano, F., Ghe, C., Isgaard, J., Papotti, M., Ghigo, E. and Muccioli, G. (2007). Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signalling. Endocrinology 148(2): 512-529.
191
Grant, D., Zhang, W., McGhee, E., Bunney, T., Talbot, C., Kumar, S., Munro, I., Dunsby, C., Neil, M. and Katan, M. (2008). Multiplexed FRET to image multiple signalling events in live cells. Biophysical Journal 95(10): 69-71.
Green, B., Irwin, N. and Flatt, P. (2007). Direct and indirect effects of obestatin
peptides on food intake and the regulation of glucose homeostasis and insulin secretion in mice. Peptides 28(5): 981-987.
Gregan, B., Juergensen, J., Papsdorf, G., Furkert, J., Schaefer, M., Beyermann, M.,
Rosenthal, W. and Oksche, A. (2004). Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. Journal of Biological Chemistry 279(26): 27679-27687.
W. and Rauh, M. (2005). Identification of ghrelin in human saliva: production by the salivary glands and potential role in proliferation of oral keratinocytes. Clinical Chemistry 51(6): 997-1006.
Gualillo, O., Lago, F. and Dieguez, C. (2008). Introducing GOAT: a target for
obesity and anti-diabetic drugs? Trends in Pharmacological Sciences 29(8): 398-401.
Guo, C., Luttrell, L. M. and Price, D. T. (2000). Mitogenic signalling in androgen
sensitive and insensitive prostate cancer cell lines. Journal of Urology 163(3): 1027-32.
Gurevich, V. and Gurevich, E. (2008a). GPCR monomers and oligomers: it takes all
kinds. Trends in Neurosciences 31(2): 74-81. Gurevich, V. and Gurevich, E. (2008b). How and why do GPCRs dimerize? Trends
in Pharmacological Sciences 29(5): 234-240. Gurevich, V. and Gurevich, E. (2008c). Rich tapestry of G protein-coupled receptor
signalling and regulatory mechanisms. Molecular Pharmacology 74(2): 312-316.
Gutierrez, J., Solenberg, P., Perkins, D., Willency, J., Knierman, M., Jin, Z., Witcher,
D., Luo, S., Onyia, J. and Hale, J. (2008). Ghrelin octanoylation mediated by an orphan lipid transferase. Proceedings of the National Academy of Sciences 105(17): 6320-6325.
Habib, F., Mason, M., Smith, P. and Stitch, S. (1979). Cancer of the prostate: early
diagnosis by zinc and hormone analysis? British Journal of Cancer 39(6): 700-704.
Hague, C., Uberti, M., Chen, Z., Hall, R. and Minneman, K. (2004). Cell surface
expression of α 1D-adrenergic receptors is controlled by heterodimerization with α 1B-adrenergic receptors. Journal of Biological Chemistry 279(15): 15541-15549.
192
Hamdan, F., Audet, M., Garneau, P., Pelletier, J. and Bouvier, M. (2005). High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based {beta}-arrestin2 recruitment assay. Journal of Biomolecular Screening 10(5): 463-475.
Hanahan, D. and Weinberg, R. (2000). The hallmarks of cancer. Cell 100(1): 57-70. Hansen, J., Hansen, J., Speerschneider, T., Lyngso, C., Erikstrup, N., Burstein, E.,
Weiner, D., Walther, T., Makita, N. and Iiri, T. (2009). Lack of evidence for AT1R/B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R/B2R heterodimer? Journal of Biological Chemistry 284(3): 1831-1839.
Hansen, J., Theilade, J., Haunso, S. and Sheikh, S. (2004). Oligomerization of wild
type and nonfunctional mutant angiotensin II type I receptors inhibits Gαq Protein signalling but not ERK activation. Journal of Biological Chemistry 279(23): 24108-24115.
Hansen, J. L. and Sheikh, S. P. (2004). Functional consequences of 7TM receptor
dimerization. European Journal of Pharmaceutical Sciences 23(4-5): 301-317.
Hanson, M. A. and Stevens, R. C. (2009). Discovery of new GPCR biology: one
receptor structure at a time. Structure 17(1): 8-14. Hanson, M. A., Cherezov, V., Griffith, M. T., Roth, C. B., Jaakola, V. P., Chien, E.
Y., Velasquez, J., Kuhn, P. and Stevens, R. C. (2008). A specific cholesterol binding site is established by the 2.8 A structure of the human ß2-adrenergic receptor. Structure 16(6): 897-905.
J. and Watts, A. (2009). Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers. Biophysical Journal 96(3): 964-973.
Harrison, C. and Van der Graaf, P. (2006). Current methods used to investigate G
protein coupled receptor oligomerisation. Journal of Pharmacological and Toxicological Methods 54(1): 26-35.
Herrick-Davis, K., Weaver, B., Grinde, E. and Mazurkiewicz, J. (2006). Serotonin 5-
HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer. Journal of Biological Chemistry 281(37): 27109-27116.
Higgins, S., Gueorguiev, M. and Korbonits, M. (2007). Ghrelin, the peripheral
hunger hormone. Annals of Medicine 39(2): 116-136. Holliday, N., Holst, B., Rodionova, E., Schwartz, T. and Cox, H. (2007). Importance
of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor. Molecular Endocrinology 21(12): 3100.
193
Holst, B., Egerod, K. L., Jin, C., Petersen, P. S., Ostergaard, M. V., Hald, J.,
Sprinkel, A. M., Storling, J., Mandrup-Poulsen, T., Holst, J. J., Thams, P., Orskov, C., Wierup, N., Sundler, F., Madsen, O. D. and Schwartz, T. W. (2009). G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction. Endocrinology 150(6): 2577-2585.
Holst, B., Egerod, K., Schild, E., Vickers, S., Cheetham, S., Gerlach, L., Storjohann,
L., Stidsen, C., Jones, R. and Beck-Sickinger, A. (2007). GPR39 signalling is stimulated by zinc ions but not by obestatin. Endocrinology 148(1): 13-20.
Holst, B. and Schwartz, T. (2006). Ghrelin receptor mutations-too little height and
too much hunger. Journal of Clinical Investigation 116(3): 637-641. Holst, B., Holliday, N., Bach, A., Elling, C., Cox, H. and Schwartz, T. (2004).
Common structural basis for constitutive activity of the ghrelin receptor family. Journal of Biological Chemistry 279(51): 53806-53817.
Holst, B. and Schwartz, T. (2004). Constitutive ghrelin receptor activity as a
signalling set-point in appetite regulation. Trends in Pharmacological Sciences 25(3): 113-117.
Holst, B., Cygankiewicz, A., Jensen, T., Ankersen, M. and Schwartz, T. (2003). High
constitutive signalling of the ghrelin receptor-identification of a potent inverse agonist. Molecular Endocrinology 17(11): 2201-2210.
Hosoda, H., Kojima, M. and Kangawa, K. (2006). Biological, physiological, and
pharmacological aspects of ghrelin. Journal of Pharmacological Sciences 100(5): 398-410.
Hosoda, H., Kojima, M., Matsuo, H. and Kangawa, K. (2000). Ghrelin and des-acyl
ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochemical and Biophysical Research Communications 279(3): 909-913.
Howard, A. D., Feighner, S. D., Cully, D. F., Arena, J. P., Liberator, P. A.,
Rosenblum, C. I., Hamelin, M., Hreniuk, D. L., Palyha, O. C., Anderson, J., Paress, P. S., Diaz, C., Chou, M., Liu, K. K., McKee, K. K., Pong, S. S., Chaung, L. Y., Elbrecht, A., Dashkevicz, M., Heavens, R., Rigby, M., Sirinathsinghji, D. J., Dean, D. C., Melillo, D. G., Patchett, A. A., Nargund, R., Griffin, P. R., DeMartino, J. A., Gupta, S. K., Schaeffer, J. M., Smith, R. G. and Van der Ploeg, L. H. (1996). A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273(5277): 974-977.
Huynh, H., Nguyen, T. T., Chow, K. H., Tan, P. H., Soo, K. C. and Tran, E. (2003).
Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: its role in tumor progression and apoptosis. BMC Gastroenterology 3: 19.
Hyman, M. and Arp, D. (1993). An electrophoretic study of the thermal-dependent
and reductant-dependent aggregation of the 27 kDa component of ammonia
194
monooxygenase from Nitrosomonas europaea. Electrophoresis 14: 619-627. Inhoff, T., Wiedenmann, B., Klapp, B., Mönnikes, H. and Kobelt, P. (2009). Is
desacyl ghrelin a modulator of food intake? Peptides 30: 991-994. Inui, A., Asakawa, A., Bowers, C., Mantovani, G., Laviano, A., Meguid, M. and
Fujimiya, M. (2004). Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. The FASEB Journal 18(3): 439-456.
Ishii, K., Otsuka, T., Iguchi, K., Usui, S., Yamamoto, H., Sugimura, Y., Yoshikawa,
K., Hayward, S. and Hirano, K. (2004). Evidence that the prostate-specific antigen (PSA)/Zn2+ axis may play a role in human prostate cancer cell invasion. Cancer Letters 207(1): 79-87.
Isik, N., Hereld, D. and Jin, T. (2008). Fluorescence resonance energy transfer
imaging reveals that chemokine-binding modulates heterodimers of CXCR4 and CCR5 receptors. PLoS One 3(10): e3424.
Jaakola, V. P., Griffith, M. T., Hanson, M. A., Cherezov, V., Chien, E. Y., Lane, J.
R., Ijzerman, A. P. and Stevens, R. C. (2008). The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322(5905): 1211-7.
Jacoby, E., Bouhelal, R., Gerspacher, M. and Seuwen, K. (2006). The 7TM G-
protein-coupled receptor target family. ChemMedChem 1(8): 760-782. James, J., Oliveira, M., Carmo, A., Iaboni, A. and Davis, S. (2006). A rigorous
experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nature Methods 3: 1001-1006.
Jeffery, P., Murray, R., Yeh, A., McNamara, J., Duncan, R., Francis, G., Herington,
A. and Chopin, L. (2005). Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocrine-related cancer 12(4): 839-850.
Jeffery, P., Herington, A. and Chopin, L. (2003). The potential autocrine/paracrine
roles of ghrelin and its receptor in hormone-dependent cancer. Cytokine and Growth Factor Reviews 14(2): 113-122.
Jeffery, P., Herington, A. and Chopin, L. (2002). Expression and action of the growth
hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. Journal of Endocrinology 172(3): 7-11.
Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T. and Thun, M. (2008).
Cancer statistics, 2008. CA: A Cancer Journal for Clinicians 58(2): 71-96. Jensen, A., Hansen, J., Sheikh, S. and Brauner-Osborne, H. (2002). Probing
intermolecular proteinprotein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET). FEBS Journal 269(20): 5076-5087.
195
Jiang, H., Betancourt, L. and Smith, R. (2006). Ghrelin amplifies dopamine
signalling by cross talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype 1 heterodimers. Molecular Endocrinology 20(8): 1772-1785.
Jones, K., Borowsky, B., Tamm, J., Craig, D., Durkin, M., Dai, M., Yao, W.,
Johnson, M., Gunwaldsen, C. and Huang, L. (1998). GABA B receptors function as a heteromeric assembly of the subunits GABA B R1 and GABA B R2. Nature 396: 674-679.
Jordan, B. A. and Devi, L. A. (1999). G-protein-coupled receptor heterodimerization
modulates receptor function. Nature 399(6737): 697-700. Kamiya, T., Saitoh, O., Yoshioka, K. and Nakata, H. (2003). Oligomerization of
adenosine A2A and dopamine D2 receptors in living cells. Biochemical and Biophysical Research Communications 306(2): 544-549.
Kanamoto, N., Akamizu, T., Tagami, T., Hataya, Y., Moriyama, K., Takaya, K.,
Hosoda, H., Kojima, M., Kangawa, K. and Nakao, K. (2004). Genomic structure and characterization of the 5'-flanking region of the human ghrelin gene. Endocrinology 145(9): 4144-4153.
Kapica, M., Zabielska, M., Puzio, I., Jankowska, A., Kato, I., Kuwahara, A. and
Zabielski, R. (2007). Obestatin stimulates the secretion of pancreatic juice enzymes through a vagal pathway in anaesthetized rats - preliminary results. Journal of Physiology and Pharmacology 58(Suppl 3): 123-30.
Katergari, S., Milousis, A., Pagonopoulou, O., Asimakopoulos, B. and Nikolettos, N.
(2008). Ghrelin in pathological conditions. Endocrine Journal 55(3): 439-453.
Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P.,
Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R., Karschin, A. and Bettler, B. (1998). GABA(B)-receptor subtypes assemble into functional heteromeric complexes. Nature 396(6712): 683-687.
Kent, T., McAlpine, C., Sabetnia, S. and Presland, J. (2007). G-protein-coupled
receptor heterodimerization: Assay technologies to clinical significance. Current Opinion in Drug Discovery and Development 10(5): 580-589.
Kenworthy, A. (2001). Imaging protein-protein interactions using fluorescence
resonance energy transfer microscopy. Methods 24(3): 289-296. Kenworthy, A. and Edidin, M. (1998). Distribution of a
glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of< 100 A using imaging fluorescence resonance energy transfer. The Journal of Cell Biology 142(1): 69-84.
Kim, M., Yoon, C., Jang, P., Park, Y., Shin, C., Park, H., Ryu, J., Pak, Y., Park, J.
196
and Lee, K. (2004). The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes. Molecular Endocrinology 18(9): 2291-2301.
Kim, S., Her, S., Park, S., Kim, D., Park, K., Lee, H., Han, B., Kim, M., Shin, C. and
Kim, S. (2005). Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells. Bone 37(3): 359-369.
Schürmann, A., Joost, H., Jandacek, R. and Hale, J. (2009). GOAT links dietary lipids with the endocrine control of energy balance. Nature Medicine 15(7): 741-745.
Kobelt, P., Wisser, A., Stengel, A., Goebel, M., Bannert, N., Gourcerol, G., Inhoff,
T., Noetzel, S., Wiedenmann, B. and Klapp, B. (2008). Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents. Peptides 29(6): 1018-1027.
Kobilka, B. and Schertler, G. F. (2008). New G-protein-coupled receptor crystal
structures: insights and limitations. Trends in Pharmacological Sciences 29(2): 79-83.
Kocan, M., See, H., Seeber, R., Eidne, K. and Pfleger, K. (2008). Demonstration of
improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells. Journal of Biomolecular Screening 13(9): 888-898.
Kohno, D., Gao, H., Muroya, S., Kikuyama, S. and Yada, T. (2003). Ghrelin directly
interacts with neuropeptide-Y-containing neurons in the rat arcuate nucleus: Ca2+ signalling via protein kinase A and N-type channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 52(4): 948-956.
Kojima, M. and Kangawa, K. (2008). Structure and function of ghrelin. Results and
Problems in Cell Differentiation 46: 89-115. Kojima, M. and Kangawa, K. (2005). Ghrelin: structure and function. Physiological
Reviews 85(2): 495-522. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H. and Kangawa, K.
(1999). Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402(6762): 656-660.
Kolakowski, L. (1994). GCRDb: a G-protein-coupled receptor database. Receptors &
Channels 2(1): 1-7. Kroeger, K., Pfleger, K. and Eidne, K. (2004). G-protein coupled receptor
oligomerization in neuroendocrine pathways. Frontiers in Neuroendocrinology 24(4): 254-278.
Kroeger, K., Hanyaloglu, A., Seeber, R., Miles, L. and Eidne, K. (2001). Constitutive
and agonist-dependent homo-oligomerization of the thyrotropin-releasing
197
hormone receptor - Detection in living cells using bioluminescence resonance energy transfer. Journal of Biological Chemistry 276(16): 12736-12743.
Krupnick, J. and Benovic, J. (1998). The role of receptor kinases and arrestins in G
protein-coupled receptor regulation. Annual Review of Pharmacology and Toxicology 38(1): 289-319.
Lagaud, G., Young, A., Acena, A., Morton, M., Barrett, T. and Shankley, N. (2007).
Obestatin reduces food intake and suppresses body weight gain in rodents. Biochemical and Biophysical Research Communications 357(1): 264-269.
Lagerström, M. and Schiöth, H. (2008). Structural diversity of G protein-coupled
receptors and significance for drug discovery. Nature Reviews Drug Discovery 7(4): 339-357.
Lander, E., Linton, L., Birren, B., Nusbaum, C., Zody, M., Baldwin, J., Devon, K.,
Dewar, K., Doyle, M. and FitzHugh, W. (2001). The International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409: 860-921.
Lanfranco, F., Baldi, M., Cassoni, P., Bosco, M., Ghé, C. and Muccioli, G. (2008).
Ghrelin and prostate cancer. Vitamins and Hormones 77: 301-324. Lau, P., Chow, K., Chan, C., Cheng, C. and Wise, H. (2009). The constitutive
activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent pathway. Molecular and Cellular Endocrinology 299: 232-239.
Lauwers, E., Landuyt, B., Arckens, L., Schoofs, L. and Luyten, W. (2006). Obestatin
does not activate orphan G protein-coupled receptor GPR39. Biochemical and Biophysical Research Communications 351(1): 21-25.
Lavoie, C., Mercier, J., Salahpour, A., Umapathy, D., Breit, A., Villeneuve, L., Zhu,
W., Xiao, R., Lakatta, E. and Bouvier, M. (2002). ß1/ß2-adrenergic receptor heterodimerization regulates ß2-adrenergic receptor internalization and ERK signalling efficacy. Journal of Biological Chemistry 277(38): 35402-35410.
Lee, H., Wang, G., Englander, E., Kojima, M. and Greeley Jr, G. (2002). Ghrelin, a
new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations. Endocrinology 143(1): 185-190.
Lee, S., So, C., Rashid, A., Varghese, G., Cheng, R., Lanca, A., O'Dowd, B. and
George, S. (2004). Dopamine D1 and D2 receptor co-activation generates a novel phospholipase C-mediated calcium signal. Journal of Biological Chemistry 279(34): 35671-35678.
Lefkowitz, R. (2007). Seven transmembrane receptors: something old, something
new. Acta Physiologica 190(1): 9-19. Lefkowitz, R. and Shenoy, S. (2005). Transduction of receptor signals by ß-arrestins.
198
Science 308(5721): 512-517. Leite-Moreira, A. and Soares, J. (2007). Physiological, pathological and potential
therapeutic roles of ghrelin. Drug Discovery Today 12(7-8): 276-288. Leung, P., Chow, K., Lau, P., Chu, K., Chan, C., Cheng, C. and Wise, H. (2007). The
truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor. Cellular Signalling 19(5): 1011-1022.
Levoye, A., Dam, J., Ayoub, M., Guillaume, J., Couturier, C., Delagrange, P. and
Jockers, R. (2006). The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization. The EMBO journal 25(13): 3012-3023.
Li, A., Cheng, G., Zhu, G. and Tarnawski, A. (2007). Ghrelin stimulates
angiogenesis in human microvascular endothelial cells: Implications beyond GH release. Biochemical and Biophysical Research Communications 353(2): 238-243.
Li, W., Gavrila, D., Liu, X., Wang, L., Gunnlaugsson, S., Stoll, L., McCormick, M.,
Sigmund, C., Tang, C. and Weintraub, N. (2004). Ghrelin inhibits proinflammatory responses and nuclear factor-kappa B activation in human endothelial cells. Circulation 109(18): 2221-2226.
Liang, J., Liu, Y., Zou, J., Franklin, R., Costello, L. and Feng, P. (1999). Inhibitory
effect of zinc on human prostatic carcinoma cell growth. The Prostate 40(3): 200-207.
Liang, Y., Fotiadis, D., Filipek, S., Saperstein, D., Palczewski, K. and Engel, A.
(2003). Organization of the G Protein-coupled receptors rhodopsin and opsin in native membranes. Journal of Biological Chemistry 278(24): 21655-21662.
Limbird, L. and Lefkowitz, R. (1976). Negative cooperativity among beta-adrenergic
receptors in frog erythrocyte membranes. Journal of Biological Chemistry 251(16): 5007-5014.
Limbird, L., Meyts, P. and Lefkowitz, R. (1975). Beta-adrenergic receptors: evidence
for negative cooperativity. Biochemical and Biophysical Research Communications 64(4): 1160-1169.
Liu, K., Zhang, W., Liu, L., He, D., Zhou, G., Zhou, L. and Hu, R. (2009). Ghrelin
inhibits apoptosis induced by high glucose and sodium palmitate in adult rat cardiomyocytes through the PI3K-Akt signalling pathway. Regulatory Peptides 155(1-3): 62-69.
Lopez-Gimenez, J., Canals, M., Pediani, J. and Milligan, G. (2007). The α1b-
adrenoceptor exists as a higher-order oligomer: Effective oligomerization is required for receptor maturation, surface delivery, and function. Molecular
199
Pharmacology 71(4): 1015-1029. Lu, Y., Cai, Z., Xiao, G., Liu, Y., Keller, E. T., Yao, Z. and Zhang, J. (2007). CCR2
expression correlates with prostate cancer progression. Journal of Cellular Biochemistry 101(3): 676-85.
Lukasiewicz, S., Blasiak, E., Faron-Gorecka, A., Polit, A., Tworzydlo, M., Gorecki,
A., Wasylewski, Z. and Dziedzicka-Wasylewska, M. (2007). Fluorescence studies of homooligomerization of adenosine A2A and serotonin 5-HT1A receptors reveal the specificity of receptor interactions in the plasma membrane. Pharmacological Reports 59(4): 379-392.
Maccarinelli, G., Sibilia, V., Torsello, A., Raimondo, F., Pitto, M., Giustina, A.,
Netti, C. and Cocchi, D. (2005). Ghrelin regulates proliferation and differentiation of osteoblastic cells. Journal of Endocrinology 184(1): 249-256.
Maggio, R., Vogel, Z. and Wess, J. (1993). Coexpression studies with mutant
muscarinic/adrenergic receptors provide evidence for intermolecular" cross-talk" between G-protein-linked receptors. Proceedings of the National Academy of Sciences 90(7): 3103-3107.
Margeta-Mitrovic, M., Jan, Y. and Jan, L. (2000). A trafficking checkpoint controls
GABAB receptor heterodimerization. Neuron 27(1): 97-106. Martini, A., Fernandez-Fernandez, R., Tovar, S., Navarro, V., Vigo, E., Vazquez, M.,
Davies, J., Thompson, N., Aguilar, E. and Pinilla, L. (2006). Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats. Endocrinology 147(5): 2374-2382.
Marullo, S. and Bouvier, M. (2007). Resonance energy transfer approaches in
molecular pharmacology and beyond. Trends in Pharmacological Sciences 28(8): 362-365.
Kojima, M. and Kangawa, K. (2000). Ghrelin stimulates gastric acid secretion and motility in rats. Biochemical and Biophysical Research Communications 276(3): 905-908.
Mawson, C. and Fischer, M. (1952). The occurrence of zinc in the human prostate
gland. Canadian Journal of Medical Sciences 30(4): 336-339. Mazzocchi, G., Neri, G., Rucinski, M., Rebuffat, P., Spinazzi, R., Malendowicz, L.
and Nussdorfer, G. (2004). Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects. Peptides 25(8): 1269-1277.
McGraw, D., Mihlbachler, K., Schwarb, M., Rahman, F., Small, K., Almoosa, K. and
Liggett, S. (2006). Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2-adrenergic receptors within a unique heterodimeric
200
complex. Journal of Clinical Investigation 116(5): 1400-1409. McKee, K., Tan, C., Palyha, O., Liu, J., Feighner, S., Hreniuk, D., Smith, R.,
Howard, A. and Van der Ploeg, L. (1997a). Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 46(3): 426-434.
McKee, K. K., Palyha, O. C., Feighner, S. D., Hreniuk, D. L., Tan, C. P., Phillips, M.
S., Smith, R. G., Van der Ploeg, L. H. and Howard, A. D. (1997b). Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Molecular Endocrinology 11(4): 415-423.
McLaughlin, J., Patterson, M. and Malik, A. (2007). Protease-activated receptor-3
(PAR3) regulates PAR1 signalling by receptor dimerization. Proceedings of the National Academy of Sciences 104(13): 5662-5667.
McNamara, J., Herington, A. and Chopin, L. (2002). Expression and function of the
Mellado, M., Rodríguez-Frade, J., Vila-Coro, A., de Ana, A. and Martínez-A, C.
(1999). Chemokine control of HIV-1 infection. Nature 400(6746): 723-724. Mercier, J., Salahpour, A., Angers, S., Breit, A. and Bouvier, M. (2002). Quantitative
assessment of ß1-and ß2-adrenergic receptor homo-and heterodimerization by bioluminescence resonance energy transfer. Journal of Biological Chemistry 277(47): 44925-44931.
Meyer, B., Segura, J., Martinez, K., Hovius, R., George, N., Johnsson, K. and Vogel,
H. (2006). FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells. Proceedings of the National Academy of Sciences 103(7): 2138-2143.
Michel, M., Wieland, T. and Tsujimoto, G. (2009). How reliable are G-protein-
coupled receptor antibodies? Naunyn-Schmiedeberg's Archives of Pharmacology 379(4): 385-388.
Mikhailova, M., Mayeux, P., Jurkevich, A., Kuenzel, W., Madison, F., Periasamy,
A., Chen, Y. and Cornett, L. (2007). Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3', 5'-cyclic adenosine monophosphate production. Molecular Endocrinology 21(9): 2178-2188.
Milligan, G. (2009). G protein-coupled receptor hetero-dimerization: contribution to
pharmacology and function. British Journal of Pharmacology(Published online ahead of print).
Milligan, G. (2008). A day in the life of a G protein-coupled receptor: the
contribution to function of G protein-coupled receptor dimerization. British
201
Journal of Pharmacology 153: S216-S229. Milligan, G. (2006). G-protein-coupled receptor heterodimers: pharmacology,
function and relevance to drug discovery. Drug Discovery Today 11(11-12): 541-549.
Milligan, G. (2004). G protein-coupled receptor dimerization: function and ligand
pharmacology. Molecular Pharmacology 66(1): 1-7. Milligan, G., Ramsay, D., Pascal, G. and Carrillo, J. (2003). GPCR dimerisation. Life
Sciences 74(2-3): 181-188. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J., Adams, J., Ikura, M. and Tsien, R.
(1997). Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388: 882-887.
Moechars, D., Depoortere, I., Moreaux, B., De Smet, B., Goris, I., Hoskens, L.,
Daneels, G., Kass, S., Ver Donck, L. and Peeters, T. (2006). Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor–knockout mouse. Gastroenterology 131(4): 1131-1141.
Mondal, M., Toshinai, K., Ueno, H., Koshinaka, K. and Nakazato, M. (2008).
Characterization of obestatin in rat and human stomach and plasma, and its lack of acute effect on feeding behavior in rodents. Journal of Endocrinology 198(2): 339-346.
Mungan, N., Eminferzane, S., Mungan, A., Yesilli, C., Seckiner, I., Can, M., Ayoglu,
F. and Akduman, B. (2008). Diagnostic value of serum ghrelin levels in prostate cancer. Urologia Internationalis 80(3): 245-248.
Murata, M., Okimura, Y., Iida, K., Matsumoto, M., Sowa, H., Kaji, H., Kojima, M.,
Kangawa, K. and Chihara, K. (2002). Ghrelin modulates the downstream molecules of insulin signalling in hepatoma cells. Journal of Biological Chemistry 277(7): 5667-5674.
Nagaya, N. and Kangawa, K. (2003). Ghrelin improves left ventricular dysfunction
and cardiac cachexia in heart failure. Current Opinion in Pharmacology 3(2): 146-151.
Nagaya, N., Miyatake, K., Uematsu, M., Oya, H., Shimizu, W., Hosoda, H., Kojima,
M., Nakanishi, N., Mori, H. and Kangawa, K. (2001a). Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. Journal of Clinical Endocrinology & Metabolism 86(12): 5854-5859.
Nagaya, N., Uematsu, M., Kojima, M., Ikeda, Y., Yoshihara, F., Shimizu, W.,
Hosoda, H., Hirota, Y., Ishida, H. and Mori, H. (2001b). Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 104(12): 1430-1435.
202
Nakazato, M., Murakami, N., Date, Y., Kojima, M., Matsuo, H., Kangawa, K. and Matsukura, S. (2001). A role for ghrelin in the central regulation of feeding. Nature 409: 194-198.
Nanzer, A., Khalaf, S., Mozid, A., Fowkes, R., Patel, M., Burrin, J., Grossman, A.
and Korbonits, M. (2004). Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. European Journal of Endocrinology 151(2): 233-240.
Neary, N., Druce, M., Small, C. and Bloom, S. (2006). Acylated ghrelin stimulates
food intake in the fed and fasted states but desacylated ghrelin has no effect. Gut 55(1): 135-135.
Nelson, G., Chandrashekar, J., Hoon, M., Feng, L., Zhao, G., Ryba, N. and Zuker, C.
(2002). An amino-acid taste receptor. Nature 416(6877): 199-202. Nelson, G., Hoon, M., Chandrashekar, J., Zhang, Y., Ryba, N. and Zuker, C. (2001).
Mammalian sweet taste receptors. Cell 106(3): 381-390. Nogueiras, R., Pfluger, P., Tovar, S., Arnold, M., Mitchell, S., Morris, A., Perez-
Tilve, D., Vazquez, M., Wiedmer, P. and Castaneda, T. (2007). Effects of obestatin on energy balance and growth hormone secretion in rodents. Endocrinology 148(1): 21-26.
Oldham, W. and Hamm, H. (2006). Structural basis of function in heterotrimeric G
proteins. Quarterly Reviews of Biophysics 39(2): 117-166. Otto, B., Cuntz, U., Fruehauf, E., Wawarta, R., Folwaczny, C., Riepl, R., Heiman,
M., Lehnert, P., Fichter, M. and Tschop, M. (2001). Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. European Journal of Endocrinology 145(5): 669-673.
Overington, J., Al-Lazikani, B. and Hopkins, A. (2006). How many drug targets are
there? Nature Reviews Drug discovery 5(12): 993-996. Overton, M. and Blumer, K. (2002). The extracellular N-terminal domain and
transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor. Journal of Biological Chemistry 277(44): 41463-41472.
Overton, M. and Blumer, K. (2000). G-protein-coupled receptors function as
oligomers in vivo. Current Biology 10(6): 341-344. Pal, R., Parker, D. and Costello, L. (2009). A europium luminescence assay of lactate
and citrate in biological fluids. Organic & Biomolecular Chemistry 7(8): 1525-1528.
Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A.,
Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M. and Miyano, M. (2000). Crystal structure of rhodopsin: A G protein-coupled
203
receptor. Science 289(5480): 739-45. Pan, W., Tu, H. and Kastin, A. (2006). Differential BBB interactions of three
ingestive peptides: obestatin, ghrelin, and adiponectin. Peptides 27(4): 911-916.
Panetta, R. and Greenwood, M. (2008). Physiological relevance of GPCR
oligomerization and its impact on drug discovery. Drug Discovery Today 13(23-24): 1059-1066.
Luton, M., Grouselle, D. and de Kerdanet, M. (2006). Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. Journal of Clinical Investigation 116(3): 760-768.
Park, J., Scheerer, P., Hofmann, K., Choe, H. and Ernst, O. (2008). Crystal structure
of the ligand-free G-protein-coupled receptor opsin. Nature 454(7201): 183-188.
Park, P. and Palczewski, K. (2005). Diversifying the repertoire of G protein-coupled
receptors through oligomerization. Proceedings of the National Academy of Sciences 102(25): 8793-8794.
Patchett, A., Nargund, R., Tata, J., Chen, M., Barakat, K., Johnston, D., Cheng, K.,
Chan, W., Butler, B. and Hickey, G. (1995). Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue. Proceedings of the National Academy of Sciences 92(15): 7001-7005.
Paul, S., Palczewski, K., Filipek, S. and Wells, J. (2004). Oligomerization of G
protein-coupled receptors: past, present, and future. Biochemistry 43(50): 15643-15656.
Pazos, Y., Casanueva, F. and Camiña, J. (2008). Basic aspects of ghrelin action.
Vitamins and Hormones 77: 89-119. Pazos, Y., Alvarez, C. J., Camina, J. P. and Casanueva, F. F. (2007). Stimulation of
extracellular signal-regulated kinases and proliferation in the human gastric cancer cells KATO-III by obestatin. Growth Factors 25(6): 373-381.
M., Lucas, P., Monterrubio, M., Martinez-A, C. and Mellado, M. (2008). Ligand stabilization of CXCR4/δ-opioid receptor heterodimers reveals a mechanism for immune response regulation. European Journal of Immunology 38(2): 537 - 549.
Pettersson, I., Muccioli, G., Granata, R., Deghenghi, R., Ghigo, E., Ohlsson, C. and
Isgaard, J. (2002). Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation. Journal of Endocrinology 175(1): 201-209.
204
Pfeiffer, M., Kirscht, S., Stumm, R., Koch, T., Wu, D., Laugsch, M., Schroder, H.,
Hollt, V. and Schulz, S. (2003). Heterodimerization of substance P and µ-opioid receptors regulates receptor trafficking and resensitization. Journal of Biological Chemistry 278(51): 51630-51637.
Pfeiffer, M., Koch, T., Schroder, H., Klutzny, M., Kirscht, S., Kreienkamp, H., Hollt,
V. and Schulz, S. (2001). Homo-and Heterodimerization of Somatostatin Receptor Subtypes: Inactivation of sst3 receptor function by heterodimerization with sst2A. Journal of Biological Chemistry 276(17): 14027-14036.
Pfleger, K., Dromey, J., Dalrymple, M., Lim, E., Thomas, W. and Eidne, K. (2006a).
Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells. Cellular Signalling 18(10): 1664-1670.
Pfleger, K., Seeber, R. and Eidne, K. (2006b). Bioluminescence resonance energy
transfer (BRET) for the real-time detection of protein-protein interactions. Nature Protocols 1(1): 337-345.
Pfleger, K. and Eidne, K. (2005). Monitoring the formation of dynamic G-protein-
coupled receptor-protein complexes in living cells. Biochemical Journal 385(3): 625–637.
Pfleger, K. and Eidne, K. (2003). New technologies: Bioluminescence resonance
energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary 6(3): 141-151.
Pierce, K., Premont, R. and Lefkowitz, R. (2002). Seven-transmembrane receptors.
Nature Reviews Molecular Cell Biology 3(9): 639-650. Piljic, A. and Schultz, C. (2008). Simultaneous recording of multiple cellular events
by FRET. ACS Chemical Biology 3(3): 156-160. Pin, J., Neubig, R., Bouvier, M., Devi, L., Filizola, M., Javitch, J., Lohse, M.,
Milligan, G., Palczewski, K. and Parmentier, M. (2007). International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers. Pharmacological reviews 59(1): 5-13.
Piston, D. and Kremers, G. (2007). Fluorescent protein FRET: the good, the bad and
the ugly. Trends in Biochemical Sciences 32(9): 407-414. Pitcher, J., Freedman, N. and Lefkowitz, R. (1998). G protein-coupled receptor
kinases. Annual Review of Biochemistry 67(1): 653-692. Prinster, S., Hague, C. and Hall, R. (2005). Heterodimerization of g protein-coupled
receptors: specificity and functional significance. Pharmacological Reviews 57(3): 289-298.
Quaynor, S., Hu, L., Leung, P., Feng, H., Mores, N., Krsmanovic, L. and Catt, K.
(2007). Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons. Molecular Endocrinology 21(12): 3062-3070.
Raj, G. V., Barki-Harrington, L., Kue, P. F. and Daaka, Y. (2002). Guanosine
phosphate binding protein coupled receptors in prostate cancer: a review. The Journal of Urology 167(3): 1458-63.
Ramsay, D., Carr, I., Pediani, J., Lopez-Gimenez, J., Thurlow, R., Fidock, M. and
Milligan, G. (2004). High-affinity interactions between human α1A-adrenoceptor C-terminal splice variants produce homo-and heterodimers but do not generate the α1L-adrenoceptor. Molecular Pharmacology 66(2): 228-239.
Ramsay, D., Kellett, E., McVey, M., Rees, S. and Milligan, G. (2002). Homo- and
hetero-oligomeric interactions between G protein coupled receptors in living cells monitored by two variants of bioluminescence energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochemical Journal 365: 429-440.
Rasmussen, S. G., Choi, H. J., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S.,
Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fischetti, R. F., Schertler, G. F., Weis, W. I. and Kobilka, B. K. (2007). Crystal structure of the human ß2 adrenergic G-protein-coupled receptor. Nature 450(7168): 383-387.
Reimer, M., Pacini, G. and Ahren, B. (2003). Dose-dependent inhibition by ghrelin
of insulin secretion in the mouse. Endocrinology 144(3): 916-921. Rice, P. L., Peters, S. L., Beard, K. S. and Ahnen, D. J. (2006). Sulindac
independently modulates extracellular signal-regulated kinase 1/2 and cyclic GMP-dependent protein kinase signalling pathways. Molecular Cancer Therapeutics 5(3): 746-54.
Rice, P. L., Beard, K. S., Driggers, L. J. and Ahnen, D. J. (2004). Inhibition of
extracellular-signal regulated kinases 1/2 is required for apoptosis of human colon cancer cells in vitro by sulindac metabolites. Cancer Research 64(22): 8148-51.
Roberts, P. and Der, C. (2007). Targeting the Raf-MEK-ERK mitogen-activated
protein kinase cascade for the treatment of cancer. Oncogene 26(22): 3291-3310.
Rocha-Sousa, A., Saraiva, J., Henriques-Coelho, T., Falcao-Reis, F., Correia-Pinto, J.
206
and Leite-Moreira, A. (2006). Ghrelin as a novel locally produced relaxing peptide of the iris sphincter and dilator muscles. Experimental eye research 83(5): 1179-1187.
Rocheville, M., Lange, D., Kumar, U., Sasi, R., Patel, R. and Patel, Y. (2000).
Subtypes of the somatostatin receptor assemble as functional homo-and heterodimers. Journal of Biological Chemistry 275(11): 7862-7869.
Rossi, F., Castelli, A., Bianco, M., Bertone, C., Brama, M. and Santiemma, V.
(2008). Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation. Peptides 29(11): 2046-2051.
Ruscica, M., Dozio, E., Boghossian, S., Bovo, G., Martos Riano, V., Motta, M. and
Magni, P. (2006). Activation of the Y1 receptor by neuropeptide Y regulates the growth of prostate cancer cells. Endocrinology 147(3): 1466-73.
Sagné, C., Isambert, M., Henry, J. and Gasnier, B. (1996). SDS-resistant aggregation
of membrane proteins: application to the purification of the vesicular monoamine transporter. Biochemical Journal 316(3): 825.
Salahpour, A. and Masri, B. (2007). Experimental challenge to a 'rigorous' BRET
analysis of GPCR oligomerization. Nature Methods 4(8): 599-600. Salahpour, A., Angers, S. and Bouvier, M. (2000). Functional significance of
oligomerization of G-protein-coupled receptors. Trends in Endocrinology & Metabolism 11(5): 163-168.
Salom, D., Lodowski, D., Stenkamp, R., Trong, I., Golczak, M., Jastrzebska, B.,
Harris, T., Ballesteros, J. and Palczewski, K. (2006). Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proceedings of the National Academy of Sciences 103(44): 16123-16128.
Samson, W., Yosten, G., Chang, J., Ferguson, A. and White, M. (2008). Obestatin
inhibits vasopressin secretion: evidence for a physiological action in the control of fluid homeostasis. Journal of Endocrinology 196(3): 559-564.
Samson, W., White, M., Price, C. and Ferguson, A. (2007). Obestatin acts in brain to
inhibit thirst. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 292(1): R637-R643.
Satake, H. and Sakai, T. (2008). Recent advances and perceptions in studies of
heterodimerization between G protein-coupled receptors. Protein and Peptide Letters 15(3): 300-308.
Sato, M., Nakahara, K., Goto, S., Kaiya, H., Miyazato, M., Date, Y., Nakazato, M.,
Kangawa, K. and Murakami, N. (2006). Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord. Biochemical and Biophysical Research Communications 350(3): 598-603.
Scheerer, P., Park, J., Hildebrand, P., Kim, Y., Krauß, N., Choe, H., Hofmann, K.
207
and Ernst, O. (2008). Crystal structure of opsin in its G-protein-interacting conformation. Nature 455(7212): 497-502.
Seifert, R. and Wenzel-Seifert, K. (2002). Constitutive activity of G-protein-coupled
receptors: cause of disease and common property of wild-type receptors. Naunyn-Schmiedeberg's Archives of Pharmacology 366(5): 381-416.
Seim, I., Collet, C., Herington, A. and Chopin, L. (2007). Revised genomic structure
of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts. BMC genomics 8(1): 298.
Seoane, L., Al-Massadi, O., Pazos, Y., Paeotto, U. and Casanueva, F. (2006). Central
obestatin administration does not modify either spontaneous or ghrelin-induced food intake in rats. Journal of Endocrinological Investigation 29(8): RC13-15.
Shakibaei, M., Schulze-Tanzil, G., de Souza, P., John, T., Rahmanzadeh, M.,
Rahmanzadeh, R. and Merker, H. J. (2001). Inhibition of mitogen-activated protein kinase kinase induces apoptosis of human chondrocytes. Journal of Biological Chemistry 276(16): 13289-13294.
Sharma, M., Benharouga, M., Hu, W. and Lukacs, G. (2001). Conformational and
temperature-sensitive stability defects of the ΔF508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments. Journal of Biological Chemistry 276(12): 8942-8950.
Shiiya, T., Nakazato, M., Mizuta, M., Date, Y., Mondal, M., Tanaka, M., Nozoe, S.,
Hosoda, H., Kangawa, K. and Matsukura, S. (2002). Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. Journal of Clinical Endocrinology & Metabolism 87(1): 240-244.
Shimada, K., Nakamura, M., Ishida, E., Kishi, M., Yonehara, S. and Konishi, N.
(2002). Contributions of mitogen-activated protein kinase and nuclear factor kappa B to N-(4-hydroxyphenyl)retinamide-induced apoptosis in prostate cancer cells. Molecular Carcinogenesis 35(3): 127-37.
Shintani, M., Ogawa, Y., Ebihara, K., Aizawa-Abe, M., Miyanaga, F., Takaya, K.,
Hayashi, T., Inoue, G., Hosoda, K. and Kojima, M. (2001). Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 50(2): 227-232.
Sibilia, V., Bresciani, E., Lattuada, N., Rapetti, D., Locatelli, V., De Luca, V., Donà,
F., Netti, C., Torsello, A. and Guidobono, F. (2006). Intracerebroventricular acute and chronic administration of obestatin minimally affect food intake but not weight gain in the rat. Journal of Endocrinological Investigation 29(11): RC31-34.
Smit, M., Vischer, H., Bakker, R., Jongejan, A., Timmerman, H., Pardo, L. and
Leurs, R. (2007). Pharmacogenomic and structural analysis of constitutive G
208
protein–coupled receptor activity. Annual Review of Pharmacology and Toxicology 47: 53-87.
Smith, R., Jiang, H. and Sun, Y. (2005). Developments in ghrelin biology and
potential clinical relevance. Trends in Endocrinology & Metabolism 16(9): 436-442.
Smith, R., Cheng, K., Schoen, W., Pong, S., Hickey, G., Jacks, T., Butler, B., Chan,
W., Chaung, L. and Judith, F. (1993). A nonpeptidyl growth hormone secretagogue. Science 260(5114): 1640-1643.
Smrcka, A., Brown, K. and Sternweis, P. (1991). Regulation of
polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251(4995): 804-807.
Soares, J. and Leite-Moreira, A. (2008). Ghrelin, des-acyl ghrelin and obestatin:
Three pieces of the same puzzle. Peptides 29(7): 1255-1270. Stangelberger, A., Schally, A. V., Varga, J. L., Zarandi, M., Cai, R. Z., Baker, B.,
Hammann, B. D., Armatis, P. and Kanashiro, C. A. (2005). Inhibition of human androgen-independent PC-3 and DU-145 prostate cancers by antagonists of bombesin and growth hormone releasing hormone is linked to PKC, MAPK and c-jun intracellular signalling. European Journal of Cancer 41(17): 2735-44.
Steinmeyer, R. and Harms, G. (2009). Fluorescence resonance energy transfer and
anisotropy reveals both hetero-and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptor. Microscopy Research and Technique 72(1): 12-21.
Storjohann, L., Holst, B. and Schwartz, T. (2008a). Molecular mechanism of Zn2+
agonism in the extracellular domain of GPR39. FEBS Letters 582(17): 2583-2588.
Storjohann, L., Holst, B. and Schwartz, T. (2008b). A second disulfide bridge from
the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39. Biochemistry 47(35): 9198-9207.
Stryer, L. (1978). Fluorescence energy transfer as a spectroscopic ruler. Annual
Review of Biochemistry 47(1): 819-846. Stryer, L. and Haugland, R. (1967). Energy transfer: a spectroscopic ruler.
Proceedings of the National Academy of Sciences 58(2): 719-726. Sun, Y., Wang, P., Zheng, H. and Smith, R. (2004). Ghrelin stimulation of growth
hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proceedings of the National Academy of Sciences 101(13): 4679-4684.
Sun, Y., Ahmed, S. and Smith, R. (2003). Deletion of ghrelin impairs neither growth
209
nor appetite. Molecular and Cellular Biology 23(22): 7973-7981. Svendsen, A., Vrecl, M., Ellis, T., Heding, A., Kristensen, J., Wade, J., Bathgate, R.,
De Meyts, P. and Nohr, J. (2008). Cooperative binding of insulin-like peptide 3 to a dimeric relaxin family peptide receptor 2. Endocrinology 149(3): 1113-1120.
Szentirmai, E. and Krueger, J. (2006). Obestatin alters sleep in rats. Neuroscience
Letters 404(1-2): 222-226. Szidonya, L., Cserzo, M. and Hunyady, L. (2008). Dimerization and oligomerization
of G-protein-coupled receptors: debated structures with established and emerging functions. Journal of Endocrinology 196(3): 435.
Takahashi, K., Furukawa, C., Takano, A., Ishikawa, N., Kato, T., Hayama, S.,
Suzuki, C., Yasui, W., Inai, K. and Sone, S. (2006). The neuromedin U-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signalling pathway as a therapeutic target for lung cancer. Cancer Research 66(19): 9408-9419.
Takaya, K., Ariyasu, H., Kanamoto, N., Iwakura, H., Yoshimoto, A., Harada, M.,
Mori, K., Komatsu, Y., Usui, T. and Shimatsu, A. (2000). Ghrelin strongly stimulates growth hormone release in humans. Journal of Clinical Endocrinology & Metabolism 85(12): 4908-4911.
Tanaka, M., Naruo, T., Nagai, N., Kuroki, N., Shiiya, T., Nakazato, M., Matsukura,
S. and Nozoe, S. (2003). Habitual binge/purge behavior influences circulating ghrelin levels in eating disorders. Journal of Psychiatric Research 37(1): 17-22.
Taub, J. S., Guo, R., Leeb-Lundberg, L. M., Madden, J. F. and Daaka, Y. (2003).
Bradykinin receptor subtype 1 expression and function in prostate cancer. Cancer Research 63(9): 2037-41.
Thompson, N., Gill, D., Davies, R., Loveridge, N., Houston, P., Robinson, I. and
Wells, T. (2004). Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 145(1): 234-242.
Tokunaga, E., Oki, E., Egashira, A., Sadanaga, N., Morita, M., Kakeji, Y. and
Maehara, Y. (2008). Deregulation of the Akt pathway in human cancer. Current Cancer Drug Targets 8(1): 27-36.
Toshinai, K., Yamaguchi, H., Sun, Y., Smith, R. G., Yamanaka, A., Sakurai, T.,
Date, Y., Mondal, M. S., Shimbara, T., Kawagoe, T., Murakami, N., Miyazato, M., Kangawa, K. and Nakazato, M. (2006). Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 147(5): 2306-2314.
Toth, P., Ren, D. and Miller, R. (2004). Regulation of CXCR4 receptor dimerization
210
by the chemokine SDF-1a and the HIV-1 coat protein gp120: a fluorescence resonance energy transfer (FRET) study. Journal of Pharmacology and Experimental Therapeutics 310(1): 8-17.
Tremblay, F., Richard, A. M., Will, S., Syed, J., Stedman, N., Perreault, M. and
Gimeno, R. E. (2009). Disruption of G protein-coupled receptor 39 impairs insulin secretion in vivo. Endocrinology 150(6): 2586-2595.
Tremblay, F., Perreault, M., Klaman, L., Tobin, J., Smith, E. and Gimeno, R. (2007).
Normal food intake and body weight in mice lacking the G protein-coupled receptor GPR39. Endocrinology 148(2): 501-506.
Tschöp, M., Wawarta, R., Riepl, R., Friedrich, S., Bidlingmaier, M., Landgraf, R.
and Folwaczny, C. (2001a). Post-prandial decrease of circulating human ghrelin levels. Journal of Endocrinological Investigation 24(6): RC19-21.
Tschöp, M., Weyer, C., Tataranni, P. A., Devanarayan, V., Ravussin, E. and Heiman,
M. L. (2001b). Circulating ghrelin levels are decreased in human obesity. Diabetes 50(4): 707-709.
Tschöp, M., Smiley, D. and Heiman, M. (2000). Ghrelin induces adiposity in
rodents. Nature 407: 908-913. Tsien, R. (1998). The green fluorescent protein. Annual Review of Biochemistry
67(1): 509-544. Uberti, M., Hague, C., Oller, H., Minneman, K. and Hall, R. (2005).
Heterodimerization with β2-adrenergic receptors promotes surface expression and functional activity of α1D-adrenergic receptors. Journal of Pharmacology and Experimental Therapeutics 313(1): 16-23.
Uberti, M., Hall, R. and Minneman, K. (2003). Subtype-specific dimerization of 1-
adrenoceptors: effects on receptor expression and pharmacological properties. Molecular Pharmacology 64(6): 1379-1390.
van der Lely, A., Tschop, M., Heiman, M. and Ghigo, E. (2004). Biological,
physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocrine Reviews 25(3): 426-457.
Venter, J., Adams, M., Myers, E., Li, P., Mural, R., Sutton, G., Smith, H., Yandell,
M., Evans, C. and Holt, R. (2001). The sequence of the human genome. Science 291(5507): 1304-1351.
Vilardaga, J., Nikolaev, V., Lorenz, K., Ferrandon, S., Zhuang, Z. and Lohse, M.
(2008). Conformational cross-talk between a2A-adrenergic and µ-opioid receptors controls cell signalling. Nature Chemical Biology 4(2): 126-131.
Vogel, S., Thaler, C. and Koushik, S. (2006). Fanciful FRET. Science's STKE
2006(331): re2.
211
Volante, M., Rosas, R., Ceppi, P., Rapa, I., Cassoni, P., Wiedenmann, B., Settanni, F., Granata, R. and Papotti, M. (2009). Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth. Journal of Pathology 218(4): 458-466.
Volante, M., Allia, E., Fulcheri, E., Cassoni, P., Ghigo, E., Muccioli, G. and Papotti,
M. (2003). Ghrelin in fetal thyroid and follicular tumors and cell lines expression and effects on tumor growth. American Journal of Pathology 162(2): 645-654.
Wajnrajch, M., Ten, I., Gertner, J. and Leibel, R. (2000). Genomic organization of
the human ghrelin gene. Journal of Endocrine Genetics 1(4): 231-234. Waldhoer, M., Fong, J., Jones, R., Lunzer, M., Sharma, S., Kostenis, E., Portoghese,
P. and Whistler, J. (2005). A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proceedings of the National Academy of Sciences 102(25): 9050-9055.
Wang, D., Hu, Y., Du, J., Hu, Y., Zhong, W. and Qin, W. (2009). Ghrelin stimulates
proliferation of human osteoblastic TE85 cells via NO/cGMP signalling pathway. Endocrine 35(1): 112-117.
Wang, J., He, L., Combs, C., Roderiquez, G. and Norcross, M. (2006). Dimerization
of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions. Molecular Cancer Therapeutics 5(10): 2474-2483.
Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov, R., Edwards, P. C.,
Henderson, R., Leslie, A. G., Tate, C. G. and Schertler, G. F. (2008). Structure of a ß1-adrenergic G-protein-coupled receptor. Nature 454(7203): 486-91.
Weigle, B., Fuessel, S., Ebner, R., Temme, A., Schmitz, M., Schwind, S., Kiessling,
A., Rieger, M. A., Meye, A., Bachmann, M., Wirth, M. P. and Rieber, E. P. (2004). D-GPCR: a novel putative G protein-coupled receptor overexpressed in prostate cancer and prostate. Biochemical and Biophysical Research Communications 322(1): 239-49.
Weikel, J., Wichniak, A., Ising, M., Brunner, H., Friess, E., Held, K., Mathias, S.,
Schmid, D., Uhr, M. and Steiger, A. (2003). Ghrelin promotes slow-wave sleep in humans. American Journal of Physiology- Endocrinology And Metabolism 284(2): 407-415.
White, J., Wise, A., Main, M., Green, A., Fraser, N., Disney, G., Barnes, A., Emson,
P., Foord, S. and Marshall, F. (1998). Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396(6712): 679-682.
Whorton, M., Jastrzebska, B., Park, P., Fotiadis, D., Engel, A., Palczewski, K. and
Sunahara, R. (2008). Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer. Journal of Biological Chemistry 283(7):
212
4387-4394. Whorton, M., Bokoch, M., Rasmussen, S., Huang, B., Zare, R., Kobilka, B. and
Sunahara, R. (2007). A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proceedings of the National Academy of Sciences 104(18): 7682-7687.
Wilson, S., Wilkinson, G. and Milligan, G. (2005). The CXCR1 and CXCR2
receptors form constitutive homo-and heterodimers selectively and with equal apparent affinities. Journal of Biological Chemistry 280(31): 28663-28674.
Wilt, T., MacDonald, R., Rutks, I., Shamliyan, T., Taylor, B. and Kane, R. (2008).
Systematic review: The comparative effectiveness and harms of treatments for clinically localized prostate cancer. Annals of Internal Medicine 148(6): 435-448.
Woehler, A., Wlodarczyk, J. and Ponimaskin, E. (2008). Specific oligomerization of
the 5-HT1A receptor in the plasma membrane. Glycoconjugate Journal Epub.
Wren, A., Seal, L., Cohen, M., Brynes, A., Frost, G., Murphy, K., Dhillo, W., Ghatei,
M. and Bloom, S. (2001). Ghrelin enhances appetite and increases food intake in humans. Journal of Clinical Endocrinology & Metabolism 86(12): 5992-5992.
Wu, P. and Brand, L. (1994). Resonance energy transfer: methods and applications.
Analytical Biochemistry 218(1): 1-13. Wurch, T., Matsumoto, A. and Pauwels, P. (2001). Agonist-independent and-
dependent oligomerization of dopamine D2 receptors by fusion to fluorescent proteins. FEBS Letters 507(1): 109-113.
Xia, Q., Pang, W., Pan, H., Zheng, Y., Kang, J. and Zhu, S. (2004). Effects of ghrelin
on the proliferation and secretion of splenic T lymphocytes in mice. Regulatory peptides 122(3): 173-178.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R. and Greenberg, M. (1995). Opposing
effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270(5240): 1326-1331.
Xu, L. L., Stackhouse, B. G., Florence, K., Zhang, W., Shanmugam, N., Sesterhenn,
I. A., Zou, Z., Srikantan, V., Augustus, M., Roschke, V., Carter, K., McLeod, D. G., Moul, J. W., Soppett, D. and Srivastava, S. (2000). PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer. Cancer Research 60(23): 6568-72.
Xu, Y., Piston, D. and Johnson, C. (1999). A bioluminescence resonance energy
transfer (BRET) system: Application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences 96(1): 151-156.
213
Yang, J., Zhao, T., Goldstein, J. and Brown, M. (2008). Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides. Proceedings of the National Academy of Sciences 105(31): 10750-10755.
Yasuda, S., Miyazaki, T., Munechika, K., Yamashita, M., Ikeda, Y. and Kamizono,
A. (2007). Isolation of Zn2+ as an endogenous agonist of GPR39 from fetal bovine serum. Journal of Receptors and Signal Transduction 27(4): 235-246.
Yeh, A., Jeffery, P., Duncan, R., Herington, A. and Chopin, L. (2005). Ghrelin and a
novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clinical Cancer Research 11(23): 8295-8303.
Yoshioka, K., Saitoh, O. and Nakata, H. (2002). Agonist-promoted heteromeric
oligomerization between adenosine A1 and P2Y1 receptors in living cells. FEBS Letters 523(1-3): 147-151.
Zaichick, V., Sviridova, T. and Zaichick, S. (1997). Zinc in human prostate gland:
Normal, hyperplastic and cancerous. International Urology and Nephrology 29(5): 565-574.
Zhang, J., Jahr, H., Luo, C., Klein, C., Van Kolen, K., Ver Donck, L., De, A., Baart,
E., Li, J. and Moechars, D. (2008a). Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39. Molecular Endocrinology 22(6): 1464.
Zhang, J., Klein, C., Ren, P., Kass, S., Ver Donck, L., Moechars, D. and Hsueh, A.
(2007a). Response to Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake". Science 315(5813): 766.
Zhang, J., Ren, P., Avsian-Kretchmer, O., Luo, C., Rauch, R., Klein, C. and Hsueh,
A. (2005). Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 310(5750): 996-999.
Zhang, M., Yuan, F., Liu, H., Chen, H., Qiu, X. and Fang, W. (2008b). Inhibition of
proliferation and apoptosis of vascular smooth muscle cells by ghrelin. Acta Biochimica et Biophysica Sinica 40(9): 769-776.
Zhang, X., Wang, W., True, L. D., Vessella, R. L. and Takayama, T. K. (2009).
Protease-activated receptor-1 is upregulated in reactive stroma of primary prostate cancer and bone metastasis. Prostate 69(7): 727-36.
Zhang, Y., Ying, B., Shi, L., Fan, H., Yang, D., Xu, D., Wei, Y., Hu, X. and Zhang,
X. (2007b). Ghrelin inhibit cell apoptosis in pancreatic ß cell line HIT-T15 via mitogen-activated protein kinase/phosphoinositide 3-kinase pathways. Toxicology 237(1-3): 194-202.
Zizzari, P., Longchamps, R., Epelbaum, J. and Bluet-Pajot, M. (2007). Obestatin
214
partially affects ghrelin stimulation of food intake and growth hormone secretion in rodents. Endocrinology 148(4): 1648-1653.
Zorrilla, E., Iwasaki, S., Moss, J., Chang, J., Otsuji, J., Inoue, K., Meijler, M. and
Janda, K. (2006). Vaccination against weight gain. Proceedings of the National Academy of Sciences 103(35): 13226-13231.