C O M M E N T A R Y
The Year In G Protein-Coupled Receptor Research
Robert P. Millar and Claire L. Newton
Medical Research Council (MRC) Human Reproductive Sciences Unit (R.P.M., C.L.N.), The Queen’s
Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom; and MRC/UCT (R.P.M.),
Group for Receptor Biology, University of Cape Town, Cape Town 7925, South Africa
I (R.P.M.) presented “The Year In G Protein-Coupled Receptor Research” at ENDO 2009. I first
described the diversity of ligands and the five families into which the approximately 800 G
protein-coupled receptors (GPCRs) are grouped, their basic structural architectures, their preeminent role in signaling, and the enormous scope for developing drugs targeted at GPCRs. I then
spoke about some of the exciting breakthroughs in solving the atomic level structures of the active
state of rhodopsin, ␤2-adrenergic, ␤1-adrenergic, and A2A-adenosine receptors. I also described studies
on the structural changes accompanying the activation of the rhodopsin family of GPCRs. From these
recent technical advances, we can anticipate that many more GPCR structures will emerge, which will
afford us greater insight into their common and unique structural features and, particularly, the
mechanisms underlying their activation. These insights will guide us in our understanding of how
GPCRs operate, both in the normal and pathological situation. Although these crystal structures are
highly informative, it is important to recognize that they represent static frozen conformations of a
single GPCR state. New biophysical techniques are therefore being utilized to facilitate the dynamic
monitoring of GPCR structural changes in relation to ligand activation. Solving of the crystal structures
of GPCRs has also presented the real possibility of using the information of the ligand-binding pocket
to allow in silico screening for novel small-molecule ligands. I then reviewed the concept of ligandinduced selective signaling of GPCRs, which is opening up new insights into more selective drug
development. The assembly of GPCRs as homo- and heterooligomers and their phosphorylation and
association with a vast array of trafficking and signal-modulating proteins are emerging as major
mechanisms underlying the functioning of GPCRs. Differential expression and recruitment of these
proteins provide a mechanism for subtle physiological regulation of cellular activity. Finally, I mentioned some of the GPCRs that have lately come to the fore as novel regulators in endocrinology. These
included fatty acid-specific GPCRs expressed in pancreatic ␤-cells and novel neuroendocrine GPCRs
regulating reproduction. (Molecular Endocrinology 24: 261–274, 2010)
e would like to thank the Year In Endocrinology
program organizers for having the vision to review
the year in G protein-coupled receptors (GPCRs) and also
the audacity to do so! There happen to have been more
than 8000 publications on GPCRs this last year and even
more than 850 reviews. Being unable to read either all of
the reviews or the primary articles, we have necessarily
been highly selective in singling out some of the exciting
developments that have emerged from GPCR research
during the last 12–18 months. We have also taken license
W
to cover some developments from late 2007 as well as
early this year and to describe some unpublished results,
which colleagues have been kind enough to share with us.
First, we will highlight some of the really exciting discoveries of the atomic level structures of GPCRs. Next,
we’ll review ligand and accessory protein modulation of
signaling of GPCRs. The regulation of GPCR membrane
expression will follow and, finally, we will mention some
of the novel GPCRs that have lately come to the fore in
endocrinology. I (R.P.M.) have asked my post-doc, Claire
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/me.2009-0473 Received November 10, 2009. Accepted November 16, 2009.
First Published Online December 17, 2009
Abbreviations: ECL, Extracellular loop; GLP1, glucagon-like peptide 1; GnIH, gonadotropin-inhibitory hormone; GPCR, G protein-coupled receptor; ICL, intracellular loop; LiSS,
ligand-induced selective signaling; NKB, neurokinin B; NMR, nuclear magnetic resonance;
TM, transmembrane.
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Newton, to assist in transforming my original talk to the
written form and to coauthor the resulting article.
Background to GPCRs
GPCRs convey the majority (⬃80%) of signal transduction across cell membranes. They are activated by diverse
ligands, which vary from single photons through ions,
odorants, amino acids, fatty acids, neurotransmitters,
peptides/polypeptides, to proteolytic enzymes, which
cleave off receptor fragments to generate an activating
ligand and adhesion molecules. All GPCRs are located
within the plasma membrane and have a common architecture consisting of seven-transmembrane (TM) domains, connected by extracellular (ECL) and intracellular
(ICL) loops. One of the very special features of GPCRs is
that they are highly druggable, to the extent that more
than one third of all current therapeutics are directed at
them. Despite this, so far only a small percentage of the
approximately 800 known and verified GPCRs have been
targeted for therapeutics. Hence, there remains enormous
scope for researchers to delineate the numerous roles of
GPCRs in basic physiology and pathophysiology and to
thereby understand and develop new treatments for
disease.
GPCRs may be classified as five major families. The
largest family, and the one we’ll be focusing on for this
article, is the Rhodopsin family, which comprises 672
family members, including 388 odorant receptors. Despite this large number, and the fact that among these only
63 are orphan receptors (i.e. ones for which ligands are
unknown), relatively few have been identified as drug
targets. Moreover, there are probably many more drug
candidates in the family as odorant receptors, which have
been assumed to be involved exclusively in the detection
of odorants, are now emerging as the target of ligands in
tissues other than the nasal neuroepithelium. For example, this year an odorant receptor, prostate-specific G protein-coupled receptor, was shown to be expressed in prostatic cancer cells, and researchers identified its ligand as a
steroid hormone, androstenone, which has potent antiproliferative effects (1).
The four other, smaller, GPCR families are the Secretin
(15 members), the Adhesion (33 members), the Glutamate (22 members), and the Frizzled/Taste (36 members).
The family of GPCRs classified as adhesion molecules is
particularly fascinating. They have a very extended N
terminus thought to be involved in cell-cell contact. There
are, as yet, no drugs targeting them, and the majority of
them remain orphan receptors. But what is becoming evident from knockout studies in Caenorhabditis elegans
and in rodents is that they are crucial in the early devel-
Mol Endocrinol, January 2010, 24(1):261–274
opment of the embryo and also appear to have a number
of physiological roles (2). There are no orphans among
the 15 Secretin family and the 11 Frizzled GPCRs, but
two thirds of the Glutamate and most of the Taste GPCRs
remain as orphans. Only seven of all these GPCRs have
been identified as drug targets.
New Atomic Level Structure of GPCRs
As previously mentioned, GPCRs have seven-TM domains connected by ICLs and ECLs. In the absence of
structural knowledge, predictions of the atomic level
structures of GPCRs were originally based on rather speculative molecular models, and later on low-resolution
two-dimensional crystal structures of rhodopsin (3, 4).
Nevertheless, this information provided insight on the
relative positioning of the TM domains and inspiration
for testing predictions of molecular interactions derived
from the molecular models. We have used the two-dimensional GnRH receptor, which is the focus of our research
and with which we are most familiar, to illustrate the
salient functional features of Rhodopsin family GPCRs
(Fig. 1). The GnRH receptor does have some unusual
features that differ from the other members of this family
(e.g. it lacks a C-terminal tail, and the TM2 asparagine
(N87) and TM7 aspartate (D319) residues, which interact,
are more frequently in reciprocal positions). Nevertheless, the GnRH receptor does have the major hallmarks of
a rhodopsin family GPCR (see Fig. 1). By developing molecular models of the GnRH receptor based on the early
rhodopsin-predicted structures, it was possible to mutate
targeted residues that were proposed to configure the receptor, bind the ligand, or be involved in receptor activation or engagement of intracellular signaling proteins (see
review in Ref. 5). For example, the putative interaction of
TM residues mentioned above was tested by mutation
and restoration of function by reciprocal mutation. Such
established interactions are then firmly fixed in refining
the molecular model. Residues conserved in all Rhodopsin family members (boxed in Fig. 1) were shown to be
critical for function, as expected. Among these, the conserved DR sequence, located at the interface between
TM3 and ICL2, (sometimes the D is substituted with another acidic residue, E) was thought to be involved in
receptor activation and indeed, mutation of D to N,
which would release R from the ionic bond between these
residues, increases the coupling efficiency (6). Confirmation that the DR ionic bond is broken in the process of
receptor activation has emerged from the recent crystal
structures (see later). GnRH-binding sites were also identified (red residues in Fig. 1) and docking of the GnRH
nuclear magnetic resonance (NMR) structure to engage
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FIG. 1. Two-dimensional representation of the human GnRH receptor. The seven-TM ␣-helical domains (boxed) are connected by three ECLs and
three ICLs. Residues in squares are ones highly conserved throughout the rhodopsin family of GPCRs. Ligand binding residues (red) and residues
thought to be important in receptor structure or binding pocket configuration (green) are shown. These include residues involved in disulfide bond
formation (black lines) and a glycosylation site. Residues involved in receptor activation are shown in blue. Residues involved in coupling to G
proteins are shown in orange. Putative protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites are indicated. The intermolecular
interactions between GnRH I residues and the receptor are indicated with red lines. Residues shown to alter selectivity for relative binding affinities
of GnRH I and GnRH II are shown in bold circles. [Modified with permission from R. P. Millar et al.: Front Neuroendocrinol 29:17, 2008 (23).
©Elsevier.]
these residues added further information for the constraints
of the GnRH receptor structure and further refinement. Although considerable progress could be made in this way, in
the absence of crystal structures, these physical structures
were essential to transform interpretation into fact.
Scientists faced a big challenge of obtaining accurate
information on the three-dimensional structure of GPCRs
to reveal the TM helical clusters surrounding the binding
pocket. Crucial to an understanding of how any protein
carries out its functions is the knowledge of the protein’s
three-dimensional structure. Such structural information
provides the necessary framework for determining mechanisms of action of proteins. In 2000, Palczewski et al. (7),
solved the structure of rhodopsin, which had a major
impact in confirming and refuting previous predictions on
the structure and function of rhodopsin. The structure
also provided more accurate information for sequence
alignment and modeling of other GPCRs. This has been
followed over the last 18 months by the solving of the structures of the ␤2-adrenergic, ␤1-adrenergic, and the A2A-adenosine receptors, as well as the structure of the unliganded
active opsin (Ops) and the active opsin bound to an 11amino acid C-terminal sequence of G␣ transducin (Ops*)
(8 –13), all of which has been key to our understanding of
the molecular mechanisms underlying GPCR activation for
transmission of signals into cells. Aside from the important
biomedical implications of solving the structures of these
receptors, the findings provide insight into understanding
how proteins in general can take up different conformations,
into their associations with other molecules and into the
signaling/activation process.
The first crystal structures that emerged after that of
rhodopsin were from Brian Kobilka’s laboratory. Solving
the ␤2-adrenergic crystal structure presented an enormous challenge in producing sufficient pure protein,
stabilizing flexible domains such as ICL3, defining appropriate detergent/lipidic environments and crystallization
conditions, and developing microdiffraction technology
to obtain x-ray data from very small crystals. Kobilka and
co-workers (10) first used an antibody to stabilize the
ICL3 (which is highly mobile and compromises crystallization) and achieved a 3.4 –3.7Å-resolution, which revealed the TM domains very clearly. This was followed
up with another crystal structure in which ICL3 was stabilized by substitution with lysozyme, a highly structured
molecule, which improved the resolution to 2.4Å so that
ECL structure could be visualized as well as elements of
the ICLs (13). The structure also shows membrane cholesterol interaction with TM domains 2, 3, and 4, which is
suggested to be the site at which small molecules can act
allosterically at GPCRs to potentially activate or inactivate them (14) (Fig. 2).
Comparison of the four solved GPCR structures reveal
two important features. The first is what we call “structural convergence” (i.e. the similarities in structure). The
second is “structural divergence” (i.e. the features that
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this highly structured closure at the top of rhodopsin,
retinol is retained within an environment where it would
not be easily hydrolyzed. Thus, the new solved structures
revealing that extracellular domains in the ␤2-adrenergic
and adenosine A2A receptors are located to one side of the
TM cluster provided a mechanism for small ligand access.
FIG. 2. Three-dimensional structure of the ␤2-adrenergic receptor.
The three-dimensional structure of the ␤2-adrenergic receptor
stabilized by lysozyme substitution of ICL3, with a resolution of 2.4Å.
The inverse agonist carazolol cocrystallized with the receptor is shown
in purple, bound to cognate residues (yellow). ECL2 is shown as a
helical structure above the TM helices. Molecules of water are shown
in red, membrane cholesterol molecules interacting with the receptor
are shown in beige, and an ICL, made up of the carboxyl terminus
tethered to the membrane by an palmitoyl residue, is shown in
orange. [Figure kindly supplied by Brian Kobilka, Department of
Molecular and Cellular Physiology, Stanford University School of
Medicine, Stanford, CA.]
differ). Figure 3A shows solved structures of four GPCRs.
It is evident from superimposition of the TM domains
that these are strikingly similar. The docked ligands
(shown in orange) are also fairly similar in occupying
much the same space, although adenosine docks somewhat more superficially (15). Thus, GPCRs tend to accommodate these small molecules with similar spatial arrangement, but the particular interactions with amino
acid side chains are quite different. The structures of the
ECL2, however, are quite divergent. In rhodopsin, for
example, Fig. 3B shows that the top of the TM cluster
seems to be occluded by the N terminus and ECL2 domains, whereas the top of the TM cluster appears to be
open in the other receptors with ECL2 positioned to one
side (15). The occlusion of the TM cluster pocket by
ECL2 presented a considerable conundrum when the
structure of rhodopsin was first solved because ligands
would not have access to the binding pocket. In forming
GPCR activation
One of the big questions in GPCR function is how does
receptor activation occur? There have been many postulates over the years and a plethora of data and speculations presented on the basis of modeling, biophysical
data, and mutagenesis studies. Thus, the three-dimensional structures solved during the past year have contributed enormously to advancing our understanding of the
molecular mechanisms involved in receptor activation.
Figure 4A beautifully demonstrates what changes occur
in the rhodopsin activation process. Opsin, which reflects
the active state of rhodopsin, is depicted in yellow helices
(9); active state opsin bound to the C-terminal peptide of
the G protein (transducin) in orange helices (11); and
inactive rhodopsin, in mauve (16). TM6, which is predicted to move, does so around 3 or 4Å in the active opsin
crystal structures. This movement allows access for the G
protein transducin to interact with helices (TMs) 6 and 5
and induction of an ␣-helical-type structure as it associates with helices 7, 6, and 5. These molecular changes are
brought about by the breaking of the ionic lock in rhodopsin comprising ionic interactions in the conserved
E/DR motif between Arg135 and the adjacent Glu134, and
with a second Glu247 in TM6 (11). The disruption of the
Arg ionic bond with its adjacent Asp acidic residue in
TM3 had been implicated by Ballesteros and colleagues
(6) in the activation of the GnRH receptor a decade ago.
The recent crystal structures of active opsin and inactive
rhodopsin clearly show that activation is accompanied by
the disruption of the Arg135/Glu134 ionic bond (Fig. 4B).
This results in the formation of a new interaction of
Arg135 with Tyr223 in TM5 (11).
Limitations of GPCR crystal structures: alternative
biophysical approaches
Although crystal structures of GPCRs have revolutionized our understanding of the operation of these receptors, they do have limitations. First, it is extremely difficult and labor intensive to produce three-dimensional
x-ray crystals; therefore, the degree to which producing
crystals of the large number of GPCRs is achievable is
uncertain and will depend upon new improved methodology. Second, these structures represent a single conformational state, yet GPCRs appear to be highly dynamic
and assume different conformational states and go
through various transitions in their interactions with li-
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FIG. 3. Structures of GPCRs solved to date. Comparison of the three-dimensional structures of rhodopsin/opsin, ␤-adrenergic, and adenosine
receptors. All structures are shown with the same orientation. TM helices are shown in purple, intracellular regions are shown in blue and
extracellular regions are shown in brown. The ligands are shown in orange, and bound lipids are shown in yellow. A, Transmembrane view of the
structures of rhodopsin/opsin, ␤2-adrenergic, ␤1-adrenergic, and adenosine A2A receptors showing similar relative positions of the TM domains
and location of the ligand-docking sites with the adenosine analog occupying a slightly more superficial position. B, Extracellular view of
rhodopsin, ␤-adrenergic, and adenosine receptor structures showing occlusion of the binding pocket by the extracellular domains for rhodopsin
but accessibility of the ligand-binding pockets for the ␤-adrenergic and adenosine receptors. [Both panels reproduced with permission from M. A.
Hanson and R. C. Stevens: Structure 17:8, 2009 (15). ©Cell Press.]
gands and intracellular signaling and modulating proteins. Most of the current crystal structures (with the exception of opsin) appear to represent an inactive or
partially active conformation, possibly because the stability of this state is greater and it is a preferred conformation for crystallization. Therefore, other biophysical techniques are needed to inform us about the dynamic
changes in GPCR structures upon their activation.
One exciting development from Wayne Hubbell’s laboratory is site-directed spin labeling of rhodopsin. This
technique involves specifically labeling the receptor with
spin labels (organic molecules able to interact with other
molecules and which contain an unpaired electron) before
analysis by electron paramagnetic resonance spectroscopy. This dynamic new technology allowed them to look
at the real-time structural movements in rhodopsin and
demonstrated that TM6 moved several angstroms on activation (17).
Another approach, used by Brian Kobilka’s group, involved substituting putative interacting amino acids of
the ␤2-adrenergic receptor with cysteine and tryptophan.
The cysteines were then labeled with the fluorophore bimane, and the quenching of the fluorescent signal by the
tryptophan was used to monitor the distance between the
two residues when the receptor is in the active (agonistbound) or inactive (inverse agonist-bound) states. When
activated with an agonist, an increase in signal occurred,
which reflected the movement of a fluorescent cysteine
residue in TM6 away from a tryptophan TM3 so that
quenching no longer occurred (18). This technique has
now been used with other residues to start to build up an
overall picture of the movements and conformational
changes in GPCRs on activation.
Yet another elegant approach, used by Brian Kobilka
and colleagues to study changes in conformation of the
␤2-adrenergic receptor, employed two-dimensional nuclear magnetic resonance (NMR) spectroscopy after systematically labeling lysine residues with 13C. This enabled
the identification of signals from specific lysines. The lysines at the intracellular face (distal TM regions and adjacent loops) were solvent exposed and mobile, whereas
K235 in ECL3 was highly constrained in accordance with
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inverse agonists. It is also intriguing that unprecedented
new classes of molecules emerged from the screen, which
were not expected to be ones that would occupy the binding site. This work is significant because it demonstrates
the proof of the concept that GPCR crystal structures can
be used for structure-based discovery of new ligands.
What’s New in GPCR Signaling?
Moving on to GPCR signaling, we’re going to highlight
some selected areas that we feel are likely to impact significantly on our understanding of the diversity of GPCR
effects on physiology and pathophysiology and how this
is going to influence drug development.
FIG. 4. Comparison of inactive and active rhodopsin conformations.
A, Comparison of the structures of active opsin (Ops), active opsin
bound to a C-terminal fragment of G␣ transducin (Ops*) (both yellow)
and rhodopsin (purple), depicting the change in position of TM6 (H6)
upon activation. [Figure kindly supplied by Richard Henderson, MRC
Laboratory of Molecular Biology, Cambridge, UK.] B, Presence of the
ionic lock between Arg135 and the adjacent Glu134 in TM3 and Glu247
in TM6. In the light-activated structure the Arg135/Glu134 ionic bond
is broken, and the distance of Glu247 from Arg135 has increased.
[Reproduced with permission from S. G. Rasmussen et al.: Nature 450:
383, 2007 (10). ©Macmillan Publishers Ltd.]
it forming a salt bridge with an aspartate residue in ECL2.
The movement of this residue was then demonstrated by
the spectral change after agonist, but not inverse agonist,
binding (65). 13C NMR spectroscopy has also been used
to show the ECL2 is displaced from the retinal binding
site upon activation of rhodopsin (19).
These fascinating insights into the molecular functioning of GPCRs represent a superb intellectual tour de force.
But do these fundamental discoveries have relevance to
solving medical problems? Do they, for example, provide
the means to develop new drugs? In a recent study from
Kolb et al. (20), 972,608 molecules were screened for
their ability to occupy the carazolol-binding site in the
␤2-adrenergic receptor crystal structure. Because carazolol is an inverse agonist, the receptor is thought to be in
the inactive conformation. From this screen, 25 of the best
fitting molecules were selected and characterized pharmacologically. Six of them had binding affinities of less than
4 ␮M [one with an inhibition constant (Ki) of 9 nM], and
five were inverse agonists. The finding is encouraging because it demonstrates the potential of in silico screening
based on crystal structure. In fact, the best selected compound is among the most effective ␤2-adrenergic receptor
Ligand-induced selective signaling (LiSS)
The first is a concept we call LiSS, whereby different
ligands selectively recruit different intracellular signaling
proteins to produce different phenotypic effects in cells
(21). The classical view of how GPCRs operate is conventionally viewed as the receptor occurring in two states: an
inactive state and an active state, which is stabilized by
agonist binding. In this active state the receptor is able to
co-opt the intracellular signaling machinery and activate
a cascade of events culminating in the physiological function of the cell. This simple bimodal switch model has
been under scrutiny for sometime. What has recently
emerged is that receptors can assume many conformations. Each of these conformations can potentially interact with a ligand in a highly selective manner. In turn, this
specific receptor conformation selectively interacts with a
specific intracellular signaling complex. By the same token the preponderance of a specific signaling complex in
a particular cell will tend to stabilize a certain receptor
conformation, thereby inducing selectivity for a certain
ligand (Fig. 5A).
The LiSS concept was originally promulgated by Terry
Kenakin (22) and is rapidly becoming a generic theme for
GPCRs. This phenomenon is referred to by different
groups using a variety of terms such as: “functional selectivity,” “biased agonism,” “ligand-selective agonism,”
“agonist-directed trafficking of signaling,” or “agonistreceptor trafficking.” It has important implications in
specific drug development and in minimizing side effects.
One example, of many that have emerged in recent years,
comes from our own studies on the effects of the two
naturally occurring GnRHs, GnRH I and GnRH II, operating through the single GnRH type I receptor. GnRH I
is much more potent in generating inositol phosphate
than in its antiproliferative effects on certain cells,
whereas GnRH II does not show much difference between
these two effects. An extreme example is a GnRH antag-
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an appropriate intracellular context. Although these challenges are substantial, we believe they will bear fruit in the
longer term efforts of GPCR drug discovery in the spin-off
benefits of reduced failure in the clinic through lack of
specificity and off-target effects.
FIG. 5. GnRH receptor ligand-induced selective signaling. A, Schematic depicting the concept of multiple active states (R1–R4) of a single GPCR (GnRH receptor) that are selective for different agonist
ligands (L1–L4). The different active receptor states are selectively coupled to different signaling complexes (SC1–SC4) that give rise to different effects in cells. [Modified with permission from R. P. Millar et al.:
Front Neuroendocrinol 29:17, 2008 (23). ©Elsevier.] B, Effects of GnRH
I, GnRH II, and GnRH antagonist (135-25) on stimulation of G␣q G proteins (inositol phosphate production) (blue) and inhibition of proliferation (red) in HEK 293 cells stably transfected with the rat GnRH receptor. [Reprinted with permission from R. P. Millar et al.: Front
Neuroendocrinol 29:17, 2008 (23). ©Elsevier.]
onist (135-25), which has no intrinsic stimulation of inositol phosphate generation but has potent antiproliferative activity (23) (Fig. 5B). We have gone on to show that
the Tyr8 of GnRH II is the main determinant of selective
antiproliferative effects (24) and identified residues in the
TM domains (25) and ECLs (26) of the GnRH receptor
that determine selectivity for ligand binding.
The LiSS concept has now been demonstrated for
many GPCRs (e.g. see Refs. 27–31) and is creating a new
level of sophistication, which challenges the dogma that
ligand engagement of a GPCR consistently elicits a specific intracellular signal. Instead, it has become increasingly clear that the nature of the ligand and the dynamically changing intracellular environment alter the flavor
of the signaling. Indeed, it appears that there is a new era
of drug discovery on our doorstep, in which screening for
novel ligands will not simply involve receptor binding
and/or the most convenient high-throughput functional
signal output but instead will screen for the appropriate
intracellular signal, which reflects the desired phenotypic
response of a cell for a disease state or pathophysiology.
Equally, appropriate cells will have to be used to ensure
GPCR signaling independent of G proteins
Another area of development over the last few years is
GPCR signaling through proteins other than G proteins.
It is becoming increasingly apparent that there are many
ways in which GPCRs can signal independently of G proteins. So much so, that a case has been made for abandoning the term “G protein-coupled receptors” and referring to them as “seven-transmembrane receptors.” The
first convincing evidence for the existence of GPCR-independent signaling came from the laboratory of Lefkowitz
and associates (32) and has since been extensively reviewed (e.g. see Ref. 33). An example is angiotensin II at
its AT1 receptor activating both ␤-arrestin and G proteins. When antagonists such as angiotensin II-receptor
blockers (losartan and valsartan) engage the binding site,
neither signal is propagated. However, another type of
antagonist (SII) does not activate the G protein pathway
but exclusively recruits ␤-arrestin and activates ERK (34).
Thus, it is imperative when developing drugs to utilize the
signaling outfit appropriate for the clinical condition
when screening small-molecule libraries. Furthermore,
the use of generic high-throughput assays, such as monitoring ␤-arrestin recruitment, have limitations in small
molecule-screening programs.
Other Mechanisms of Modulating
GPCR Function
As discussed earlier, ligand-induced selective signaling
can be accomplished through single GPCRs recruiting
different G proteins and also non-G protein-signaling
molecules (i.e. G protein-independent signaling). The dynamic regulation of the relative preponderance of these
cell membrane and intracellular molecules will therefore
modulate the flavor of the signaling. Similarly, variations
in cellular proteins that influence the expression of
GPCRs, their translation and trafficking to the cell membrane, and their internalization will have marked effects
on ligand stimulation of cells. As summarized in Fig. 6,
the first levels of GPCR regulation are gene transcription,
translation, and posttranslational processing, which may
be regulated by the ligand itself and by other hormones
and factors. The synthesized receptor may then be regulated by other sets of proteins in its trafficking to the
membrane of the cell. On arrival at the cell membrane, the
GPCR may further associate with numerous membrane
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FIG. 6. Potential mechanisms for regulation of GPCRs. Schematic
describing how GPCRs can be regulated at many levels: from their
biosynthesis (gene transcription, translation, and posttranslational
processing) through their trafficking to the cell membrane and, once at
the cell surface, through oligomerization and interactions with various
other nonreceptor proteins or through modifications such as
differential phosphorylation.
and intracellular proteins that will potentially alter ligand
affinity, ligand selectivity, signaling, cytoskeletal and extracellular matrix interactions, and receptor internalization. GPCRs may further undergo homo- or heterooligomerization to induce transactivation of other receptors
or lead to signal modification. GPCR phosphorylation,
acetylation, palmityolation, ubiquitinylation, and myristoylation also modify receptor functional properties.
Clearly, the integrated effects of all these possibilities are
vast. New developments in the effects of phosphorylation
or oligomerization have been selected for description in
this section, and regulators of membrane trafficking will
be touched on in the next section.
Differential receptor phosphorylation
The effects of differential receptor phosphorylation on
signaling events has been reviewed by Tobin et al. (35).
Using the M3 muscarinic receptor as an example, they
demonstrated characteristic fingerprints of receptor
phosphorylation in different cells, each with its own spectrum of kinases (Fig. 7A). Each fingerprint imparts both a
different flavor of signaling and a different phenotype in
cells. The M3 receptor has also been mutated so that certain types of phosphorylation cannot take place, and
knock-in mice have been created with associated differential phenotypes, demonstrating the importance of differential phosphorylation in regulation of cellular responses to GPCR activation (35). This represents an
interesting level of regulation and opens up many possibilities for differential phosphorylation of GPCRs, which
will be affected by expression, activation, deactivation,
and recruitment of kinases and phosphatases, all of which
influence their conformation and their ability to recruit
and activate intracellular signaling pathways (Fig. 7B).
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Receptor oligomerization
Receptor oligomerization has attracted a great deal of
attention in the last few years. In a recent publication by
Ferré et al. (36), a conceptual framework was laid out to
classify the various types of oligomerization and their
resultant effects on receptor function. As summarized in
Fig. 8, they suggest that receptors that are inactive in
binding or signaling as monomers, but which become
active as oligomers, should be known as “homomeric/
heteromeric receptors,” whereas receptors that are intrinsically active as monomers but have new activities
as oligomers should be designated as “receptor homomers/
heteromers.”
Oligomerization of GPCRs can influence their signaling in several ways. For example, as shown in Fig. 9,
dopamine D1 and D2 receptors, as monomers, signal
through G␣s and G␣i, respectively, but D1/ D2 heteromers
recruit a different signaling pathway, G␣q (37). Interestingly, dopamine signaling in the brain is predominantly
through G␣q, suggesting that D1/D2 heteromers predominate in vivo (38).
Another way in which receptor oligomerization can
affect receptor function is through a process known as
“transactivation” in which oligomerization of two defective receptors is able to restore receptor functionality. For
example, it has been shown that coexpression of two mutant LH receptors in cells (one which cannot signal but
can bind LH and a second that can signal but cannot bind
LH) allows the receptors to compliment each other so that
LH is bound and receptor signaling is restored (39). These
findings have now been taken further by Rivero-Müller et
al., who have provided the only direct evidence of GPCR
oligomerization in vivo by creating knock-in mice with
each of these mutant receptors, both of which show
highly regressed reproductive tracts (Rivero-Müller,
A., personal communication). However, crossing the
two knock-in strains to produce offspring with one
allele of each mutant receptor restores reproductive
activity. This is a most elegant and singular demonstration that receptor oligomers are formed in vivo.
What are the potential future prospects and implications for research in GPCR oligomerization? All indications are that receptor oligomerization will become an
important area of study for understanding the biology
and pathophysiology of disease, and that the phenomenon offers novel therapeutic possibilities. A good example
is that of rimonabant, a cannabinoid receptor 1 antagonist/
inverse agonist, which was originally developed as an antagonist of nicotine addiction and therefore an aid to stop
smoking. The drug was found to be very effective in diminishing the urge to smoke, but a desirable side effect of
treatment was observed in appetite suppression. Research
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269
These are just a few examples of
GPCR heteromers. The formation of
heteromers between GPCRs and other
receptors or nonreceptor proteins suggests that these interactions create a
huge spectrum of additional subtleties
in the way that GPCR activities may be
modulated and how they may be involved in cross talk with other regulatory systems.
Regulation of GPCR
Membrane Expression
Understanding the mechanisms and
identifying the proteins involved in receptor internalization, down-regulation, and uncoupling from signaling
proteins has been an area of intensive
research for many years. An emerging
area of considerable interest is the identification and delineation of the roles of
proteins involved in escorting GPCRs
through the various cellular compartments to the cell surface. GPCRs are dynamically regulated to adapt responses
of cells to cognate ligands by regulating
receptor trafficking to the cell surface
or internalization and degradation/
FIG. 7. Differential GPCR phosphorylation. A, Schematic describing how differential
recycling of receptors. These processes
receptor phosphorylation can lead to differential signaling outcomes. A single GPCR subtype
therefore represent novel means of
that is able to be phosphorylated by three different protein kinases at distinct sites on the
physiological regulation and targets for
C-terminal tail and ICL3 is shown. These hypothetical protein kinases are employed in
different combinations resulting in different phosphorylation profiles. This translates to a
potential therapeutic intervention (42).
code that directs the signaling outcome of the receptor. Thus, the same receptor subtype
It is not possible to describe all these
expressed in three different tissues (tissues A–C) can show different signaling outcomes
proteins here but of particular interest is
based on differential receptor phosphorylation. [Reprinted with permission from A. B. Tobin
a group of proteins, listed in Fig. 10,
et al.: Trends Pharmacol Sci 29:413, 2008 (35). ©Elsevier.] B, Schematic describing potential
mechanisms mediating differential GPCR phosphorylation. The phosphorylation of receptors
called “nonclassical private GPCR
within different cell types can potentially be regulated by several factors, including the
chaperones,” which interact with
expression, activation/deactivation, and recruitment of different kinases and phosphatases.
GPCRs in intracellular compartments
Each level of regulation will itself be under the control of different regulatory inputs. All
these factors may result in differential phosphorylation of receptors, leading to an alteration
to facilitate their cell-surface expression
in their conformational activity and subsequent differential ligand/signaling selectivity.
(43). One such example, melanocortin
2-receptor accessory protein (MRAP),
revealed that cannabinoid receptor 1 dimerizes with the
appetite-stimulating orexin-1 receptor and that rimon- is an excellent example of the importance of these proabant antagonizes orexin A stimulation of ERK 1/2 teins. Genetically inherited hypocortisolism in patients
through the orexin-1 receptor (40). Another example is with normal ACTH and melanocortin-2 (MC2) receptor
␦-opioid/␮-opioid receptor heteromers. ␦- And ␮-opioid re- genes presented a clinical conundrum until mutations in
ceptor interactions have been implicated in ␮-opioid recep- MRAP were identified that ablated its ability to shuttle
tor-mediated morphine tolerance and physical dependence. the MC2 receptor to the surface of the cell (44 – 46).
A hybrid bivalent molecule consisting of ␮-opioid receptor Given the large number of intracellular proteins involved
agonist coupled with a ␦-opioid receptor antagonist, which in regulating GPCR trafficking to the cell surface, it is
targets these heteromers, has the potential to convey anal- evident that there are many opportunities for understandgesia without generating these unwanted side effects (41).
ing how cell surface of expression of GPCRs is regulated
270
Millar and Newton
The Year In GPCR Research
FIG. 8. Receptor oligomers: definitions and conceptual framework.
Ferré et al. (36) have proposed the following revised definitions for
oligomeric receptor nomenclature: homomeric/heteromeric receptors
are oligomers comprised of two or more receptor molecules that are
nonfunctional as monomers but are active as oligomers; receptor
homomers/heteromers are oligomers comprised of two or more
(functional) receptor molecules, which results in novel biochemical
properties distinct from those of the individual monomers. X, Inactive;
公, active.
and how this may be modulated for therapeutic interventions. For example, inhibiting MRAP function could potentially be used as an adjunct therapy for Cushing’s disease,
because the overproduction of ACTH could be counteracted by the absence of MC2 receptor expression on the cell
surface. The importance of MRAP, and its paralog MRAP2,
in the regulation of melanocortin receptors has been recently
reviewed, in detail, by Webb and Clark (47).
Pharmacochaperones: rescue of mutant and
underexpressing GPCRs
A relatively new area of GPCR pharmacology is that of
pharmacochaperones. These are hydrophobic small molecules that can penetrate the cell membrane, bind to the
nascent GPCR, and rescue or shuttle mutant and underexpressing GPCRs to the cell membrane. Conn and
Janovick (48) have pioneered the use of cell-permeant
small molecule GnRH receptor antagonists to rescue
poorly expressed GnRH mutant receptors. In our own
laboratory, we routinely employ such antagonists to facilitate the characterization of GnRH receptor mutations
directed at delineating receptor function. As an example,
a mutant human GnRH receptor, which exhibits no ino-
FIG. 9. Signal switching by dopamine receptor heteromers. Lee et al.
(37) have demonstrated that, when individually expressed, dopamine
D1 and D2 receptors are activated by the agonist SKF 83959 they signal
through coupling to G␣s and G␣i, respectively. However, SKF 83959activated D1/D2 receptor heteromers couple to G␣q, which equates to
in vivo signaling.
Mol Endocrinol, January 2010, 24(1):261–274
FIG. 10. Nonclassical private GPCR chaperones. A list of nonclassical
chaperones and escorts that regulate GPCR translocation to the
plasma membrane (43).
sitol phosphate response to GnRH, exhibits a robust
response to GnRH after preincubation with a small
molecule GnRH antagonist from Neurocrine Bioscience
(NBI42902) (Tello, J. A., unpublished data) (Fig. 11A).
Similar results have been observed using a mutant human
LH receptor, which was identified in patients with impaired fertility and has been shown to be retained intracellularly (49). When exposed to a cell-permeant small molecule agonist the mutant receptors were recruited to the cell
surface (Fig. 11B). In addition, treating the mutant receptor
with LH elicits a very poor response compared with the wild
type, whereas treatment with the small molecule agonist
restores functionality (Newton, C. L., unpublished data).
There are now quite a few examples of the rescue of underexpressing mutant GPCR endocrine receptors (50).
Novel GPCRS and Ligands
in Endocrinology
A number of new roles for GPCRs and their ligands have
been identified over the past 18 months. We have selected
examples of novel regulators of pancreatic ␤-cell function
and novel GPCRs regulating gonadotropic hormones.
Novel pancreatic ␤-cell GPCRs
About 20 GPCRs have now been shown to be expressed in pancreatic ␤-cells, all of which can potentially
stimulate or inhibit insulin secretion. The glucagon-like
peptide 1 (GLP1) receptor is one of these, and GLP1 receptor activators are already being used clinically for the
treatment of type 2 diabetes (51). Insulin secretion is stimulated by glucose transport through the glucose transporter 2 into the ␤-cell. Glucose metabolism generates
ATP which leads to the closing of potassium channels,
depolarization, and opening of voltage-gated calcium
channels. Calcium entry into the cell elicits the exocytosis
of insulin. Activation of GPCRs such as GLP1 can en-
Mol Endocrinol, January 2010, 24(1):261–274
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271
FIG. 12. Differential thiazolidinedione activation of GPR40. Different
thiazolidinedione drugs provide distinct kinetics and efficacy of ERK1/2
MAPK phosphorylation. Cells expressing GPR40 were treated with a range
of thiazolidinedione drugs [rosiglitazone (100 ␮M), ciglitazone (100 ␮M),
troglitazone (10 ␮M), and pioglitazone (100 ␮M)], and the phosphorylation
of ERK1/2 MAPK was measured over a 3-h period. [Adapted with
permission from N. J. Smith et al.: J Biol Chem 284:17527, 2009 (55).
©American Society for Biochemistry and Molecular Biology.]
FIG. 11. Rescue of mutant GPCRs using pharmacochaperones.
A, Rescue of mutant GnRH receptors by the small-molecule
pharmacochaperone NBI42902. Cells expressing mutant (E90K)
human GnRH R1 were preincubated in the presence/absence of
NBI42902 (1 ␮M) for 1 h before measurement of inositol phosphate
accumulation in response to stimulation with GnRH I. In the absence
of the small molecule no inositol phosphate production in measured,
but a robust response occurs after preincubation with the
pharmacochaperone. [Figure kindly provided by Javier Tello, MRC
Human Reproductive Sciences Unit, Edinburgh, UK (unpublished
data)]. B, Rescue of mutant LH receptors by a small-molecule
pharmacochaperone. Cells expressing mutant (S616Y) human LH
receptor (kindly supplied by Deborah Segaloff, Department of
Molecular Physiology and Biophysics, The University of Iowa, Iowa City,
IA) were preincubated in the presence/absence of a small-molecule
agonist for 24 h, before permeabilization of the cells, labeling of the
receptors with fluorescent (green) antibodies, and visualization by
confocal microscopy. In the absence of the small-molecule receptor,
localization is largely intracellular. However, preincubation with the
pharmacochaperone causes translocation of the receptor to the cell
surface as is seen in untreated wild-type human LH receptors
(Newton, C. L., unpublished data).
hance the amount of intracellular calcium, for example
through activation of G␣q/11 and subsequent generation
of inositol triphosphate and release of calcium from intracellular stores, thereby potentiating glucose stimulation of insulin secretion. Among the other GPCRs identified in ␤-cells are the newly discovered free fatty acid
receptors, GPR40, 43, and 41. GPR40 couples to G␣q/11,
so we might predict that free fatty acid would enhance the
calcium response of the ␤-cell to glucose and increase
insulin secretion. And indeed, this appears to be the case.
Insulin responses to glucose are improved in mutant mice
overexpressing the GPR40 receptor and in normal rats
treated with GRP40 agonists (52–54). Intriguingly, Milligan and colleagues (55) have shown that thiazolidinedione compounds (glitazones) used in the treatment of type
2 diabetes, are able to activate GPR40 through the recruitment of G␣q/11 leading to ERK 1/2 MAPK activation. The
various glitazones tested revealed different effective doses
(ED50) and time frames of ERK1/2 activation (Fig. 12).
These observations suggest that part of the action of glitazones may be through GPR40 and that this might underlie
variations in the clinical effectiveness of different glitazones.
Novel neuroendocrine GPCRs
regulating reproduction
There have been a number of breakthroughs in neuroendocrinology in the last year. Two of these are briefly
FIG. 13. NKB and GnIH: novel regulators of gonadotropin in man.
Schematic describing the potential mechanisms for control of
gonadotropin secretion by NKB and GnIH. Kisspeptin (Kiss) released
from kisspeptin neurons within the hypothalamus is known to
control secretion of GnRH from GnRH neurons through interaction
with its cognate receptor (KissR/GPR54). Secretion of GnRH from
these neurons then leads to secretion of gonadotropins (LH/FSH)
from pituitary gonadotropes. NKB is colocalized in kisspeptin
neurons and is postulated to be a novel regulator of GnRH secretion
either through direct interaction with GnRH neurons or through
autocrine interactions with kisspeptin neurons. GnIH is a potent
inhibitor of GnRH stimulation of gonadotropin secretion from
cultured gonadotropes but may also operate by inhibiting the
activity of GnRH neurons.
272
Millar and Newton
The Year In GPCR Research
touched on here. After the seminal discovery that kisspeptin/GPR54 acts as a major whole-body sensor mediating
diverse effects on the GnRH neuron (56 –58), Topaloglu et
al. (59) described mutations in neurokinin B (NKB) and its
receptor (TACR3), which give rise to hypogonadotropic
hypogonadism and pubertal failure in four Turkish families. How does this relate to the kisspeptin/GPR54 system? Interestingly, NKB colocalizes to kisspeptin neurons,
along with Dynorphin A, in ewes (60), so we now have an
enrapturing challenge to understand the interplay of these
three neuropeptides in modulating the GnRH neuron
through their cognate GPCRs. Are they being cosecreted
and interacting at the level of the GnRH neuron or are
they working in an autocrine feedback type of modality by
modulating each others secretion in kisspeptin neurons, or
possibly a combination of both (Fig. 13)? Their relative biosynthesis, processing, and secretion may also be differentially regulated by various neuronal inputs, providing a further level for subtlety of regulation.
Another discovery, that of a gonadotropin-inhibitory
hormone (GnIH) in birds some years ago, has gathered
momentum recently with studies in mammals. In the laboratory of Clarke et al. (61), GnIH (like kisspeptin, an
RF-amide neuropeptide) was found to be a very potent
inhibitor of GnRH stimulation of gonadotropin secretion, in cultured ovine gonadotropes and in vivo. GnIH
may also operate by directly or indirectly inhibiting the
GnRH neurons themselves (62– 64).
The discovery of NKB, dynorphin A, and GnIH as neuroendocrine regulators has provided new opportunities
for research on novel GPCRs in fine tuning the hypothalamic-pituitary-gonadal axis and provides new pathways
in which to interrogate feedback mechanisms and metabolic, photoperiod, and behavioral influences on the reproductive system (Fig. 13).
Conclusion: What Lies Ahead?
The pioneering technical advances recently accomplished
are promising to facilitate the crystallization of additional
GPCRs and the solving of their structures. Nevertheless,
the challenges remain formidable, and the structural determination of substantial numbers of GPCRs is not very
likely in the near future. Solving additional GPCR structures, and especially those of receptors from other GPCR
families, and receptors for other classes of ligands, will
provide insights into common and unique structural features involved in ligand binding, receptor activation, and
interactions with signaling proteins. This information, in
turn, will assist in guiding molecular modeling of unsolved GPCR structures more accurately.
Mol Endocrinol, January 2010, 24(1):261–274
The limitations of crystal structures in representing a
singular nondynamic state or conformation of GPCRs
may defy satisfactory resolution of the full repertoire of
events underlying GPCR function. The development of
novel biophysical techniques for monitoring these structural changes is encouraging and has considerable promise for future studies.
The insights into GPCR structure and function also
appear to be bringing the holy grail of in silico discovery
and development of small-molecule analogs closer to reality. Further finessing of the approach and translation
into success in drug discovery is now a very reasonable
expectation.
The discovery of a multitude of intracellular protein interactions with GPCRs points to almost limitless potential
for modulation of their expression, ligand selectivity, and
signaling to produce an array of cellular and physiological
phenotypes. A particular challenge will be the determination
of the actual in vivo functioning of GPCRs and the development of specific therapeutics targeting specific phenotypes.
However, the recognition of the diversity of cell contextdriven function provides the first steps toward more specific
drug development.
We can also anticipate that the generation of knockout
and hypomorph animals for every GPCR, and the discovery of GPCR ligand analogs and modulators of GPCR
function, will provide early preclinical tools for delineating physiological function and regulation.
Deorphanization of GPCRs is advancing at pace, and the
relatively under-explored large group of odorant receptors
and smaller GPCR families offer exciting possibilities to
discover new regulators of the endocrine system.
Acknowledgments
We thank Graeme Milligan, Brian Kobilka, Craig McArdle,
Richard Henderson, Scott Struthers, Zhi-liang Lu, Adam Pawson,
Antonia Roseweir, and Javier Tello for helpful input into the
presentation.
Address all correspondence and requests for reprints to: R. P.
Millar, Medical Research Council Human Reproductive Sciences
Unit, The Queen’s Medical Research Institute, Edinburgh EH16
4TJ, Scotland, United Kingdom. E-mail: [email protected].
Disclosure Summary: The authors have nothing to disclose.
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