195
The Structural Basis of G Protein
Coupled Receptor Signaling
Nobel Lecture, December 8, 2012
by Brian Kobilka
Stanford University School of Medicine, Department of Molecular and
Cellular Physiology, 279 Campus Drive, Stanford, CA 94305, USA.
Introduction
Complex organisms require a sophisticated communication network to maintain
homeostasis. Cells from different parts of our bodies communicate with
each other using chemical messengers in the form of hormones and neurotransmitters.
Cells process information encoded in these chemical messages
using G protein coupled receptors (GPCRs) located in the plasma membrane.
GPCRs are also mediate communication with the outside world. The senses of
sight, smell and taste are mediated by GPCRs. G protein coupled receptors (GPCRs)
are nature’s most versatile chemical sensors. There are over 800 GPCRs in
the human genome and they respond to a broad spectrum of chemical entities
ranging from photons, protons and calcium ions, and small organic molecules
(including odorants and neurotransmitters), to peptides and glycoproteins.
The classical role of a GPCR is to detect the presence of an extracellular agonist,
transmit the information across the plasma membrane, and activate a cytoplasmic
heterotrimeric G protein, leading to modulation of downstream effector
proteins. Taking the human β2AR as an example, binding of adrenaline leads
to activation of Gαs, stimulation of adenylyl cyclase, cAMP accumulation, PKA
activation, and phosphorylation of proteins involved in cell metabolism (Fig. 1).
However, a wealth of research has now demonstrated that many GPCRs have
more complex signaling repertoires. For example, the β2AR couples to both Gαs
and Gαi in cardiac myocytes (Xiao et al. 1999), and can also signal through MAP
kinase pathways in a G protein-independent manner via arrestin (Azzi et al. 2003;
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196 The Nobel Prizes
Shenoy et al. 2006). Similarly, the process of GPCR desensitization involves multiple
pathways, including receptor phosphorylation events, arrestin-mediated
internalization into endosomes, receptor recycling and lysosomal degradation.
These activities are further complicated by factors such as GPCR oligomerization
(Terrillon and Bouvier 2004), localization to specific membrane compartments,
and resulting differences in lipid bilayer composition. Such multifaceted functional
behavior has been observed for many different GPCRs.
How does this complexity of functional behavior reconcile with the biochemical
and biophysical properties of GPCRs? The effect of a ligand on the
structure and biophysical properties of a receptor, and thereby the biological
response, is known as the ligand efficacy. Natural and synthetic ligands can be
grouped into different efficacy classes (Fig. 1 inset): full agonists are capable of
maximal receptor stimulation; partial agonists are unable to promote full activity
even at saturating concentrations; neutral antagonists have no effect on signaling
activity, but can prevent other ligands from binding to the receptor; inverse
agonists reduce the level of basal or constitutive activity below that of the unliganded
receptor. For GPCRs capable of coupling to multiple signaling systems,
Figure 1.  The complex signaling and regulatory behavior of the β2AR. The inset illustrates
the concept of ligand efficacy.
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The Structural Basis of G Protein Coupled Receptor Signaling197
specific ligands can have different relative efficacies towards the different pathways.
In the extreme case, even opposite activities towards different signaling
pathways are observed: for the β2 adrenergic receptor (β2AR), a partial agonist
toward the arrestin/MAP kinase pathway are also inverse agonists for the classical
Galphas/cAMP/PKA pathway (Azzi et al. 2003; Wisler et al. 2007).
Given the central role played by GPCRs in nearly all physiological processes,
they represent the largest group of targets for drug discovery for a broad spectrum
of diseases. A better understanding of the structural basis for the complex
signaling behavior of GPCRs should lead to more efficient and economical approaches
to drug discovery.
Early insights into GPCR structure
First insights into GPCR structure came from the sequencing of rhodopsin and
cloning of the β2AR and other GPCRs in the 1980s. My research career in this
field began in 1984 when I joined the laboratory of Dr. Robert Lefkowitz. As a
postdoctoral fellow in the Lefkowitz laboratory, I was involved in the cloning
of the β2AR (Dixon et al. 1986). This was the first look at the primary amino acid
sequence of a GPCR activated by a diffusible ligand (hormone or neurotransmitter).
At the time we were surprised by the sequence homology with rhodopsin.
However, this comparison and insights from the cloning of other GPCRs that
soon followed confirmed the seven transmembrane topology to be a signature
of GPCRs.
My first efforts to understand the structural basis of β2AR function took advantage
of having access to other adrenergic receptors that we had cloned.
Generating chimeric receptors composed of different combinations of sequence
from β2AR and α2AAR allowed us to identify domains involved in ligand
binding and G protein coupling specificity (Kobilka et al. 1988). After I started
my lab at Stanford University at the end of 1989 we continued to refine the
map of functional domains through a series of studies using chimeric receptors
and site-directed mutants (Suryanarayana et al. 1991; Guan et al. 1992; Suryanarayana
and Kobilka 1993) these studies also identified intramolecular contacts
that helped define the arrangement of transmembrane segments in the lipid
bilayer (Mizobe et al. 1996).
Realizing the limitations of mutagenesis to define protein structure, I began
to explore methods for the large-scale production and purification of the
β2AR to enable the use of biophysical methods to study receptor structure and
the conformational changes involved in receptor activation. The β2AR was an
ideal model system for these studies because of the existing wealth of structural
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198 The Nobel Prizes
information from mutagenesis studies and the rich diversity of commercially
available ligands for this receptor (agonists, partial agonists, neutral antagonists,
and inverse agonists). Nevertheless, this effort was particularly challenging because
of the inherent problems associated with expression and purification of
these relatively unstable membrane proteins.
Initial work focused on understanding β2AR biosynthesis in an effort to
identify factors that might facilitate large-scale production (Kobilka 1990). The
β2AR is a type IIIb membrane protein and lacks a cleavable signal sequence. We
found that insertion of a cleavable signal sequence from influenza hemaglutinin
improved insertion of the amino terminus and transmembrane segment (TM)
1, and enhanced functional expression in insect cells by two-fold (Guan et al.
1992). Using this modification, together with affinity tags at the amino and carboxyl
terminus, we established a protocol to express and purify sufficient quantities
of β2AR for biophysical studies (Kobilka 1995).
In 1993, Ulrik Gether and Sansan Lin joined the lab and began applying fluorescence
spectroscopy and other biochemical and biophysical approaches to
characterize β2AR structure and conformational changes in response to binding
of various ligands. Using relatively simple techniques such as circular dichroism
and intrinsic tryptophan fluorescence gave us insights into the biochemical
behavior of the β2AR that would ultimately be important for crystallography,
such as the stabilizing effect of ligands, particularly antagonists and inverse agonists
(Lin et al. 1996). By labeling the β2AR with small, environmentally sensitive
fluorescence probes we were able to observe structural changes in response to
agonist binding (Gether et al. 1995; Gether et al. 1997; Gether et al. 1997).
These initial studies led to a series of experiments using fluorescence spectroscopy
to characterize the mechanism of agonist binding and activation.
These experiments focused primarily on transmembrane segment (TM) 6 which
earlier experiments suggested underwent the largest structural changes upon
agonist binding. Purified β2AR was labeled at the cytoplasmic end of TM6 with
a small environmentally sensitive fluorescent probe. By monitoring changes in
the fluorescence as a function of time, we observed that the agonist binding
and activation occurred through at least one conformational intermediate, and
that agonists and partial agonists stabilize distinct states (Ghanouni et al. 2001;
Swaminath et al. 2004; Swaminath et al. 2005). We also observed that agonists
alone do not stabilize a single active conformation (Ghanouni et al. 2001). As a
result of these findings, together with a growing body of evidence for ligandspecific
signaling behavior in cells, GPCRs were no longer thought to behave
as simple two-state switches. Rather, they are more properly thought of as molecular
“rheostats,” able to sample a continuum of conformations with relatively
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The Structural Basis of G Protein Coupled Receptor Signaling199
closely spaced energies (Deupi and Kobilka 2007; Deupi and Kobilka 2010).
These biophysical and functional experiments suggested that chemical interactions
between a ligand and a receptor led to the stabilization of a ligand-specific
conformation or ensemble of conformations that interact with specific cytoplasmic
signaling and regulatory proteins.
Crystallography
The first insights into the three dimensional structure of GPCRs came from rhodopsin,
which differs from most other GPCRs in its relatively high biochemical
stability and its natural abundance in a native tissue, bovine retina. Gebhard
Schertler’s lab provided the first structures of rhodopsin from two-dimensional
crystals generated in lipids from rod outer segment membranes (Unger et al.
1997). This structure revealed the general architecture of the seven transmembrane
(TM) helices, and was the basis for most GPCR homology models until
Okada and Palczewski (Palczewski et al. 2000) obtained the first high-resolution
three-dimensional structure of rhodopsin in 2000. The elegantly simple
approach developed by Okada for purifying rhodopsin from rod outer segments
using only detergent extraction suggested that lipids extracted with
rhodopsin might be important for protein stability and/or crystallogenesis
(Okada et al. 2000). More recently, Ernst and Hoffman produced the first active
state structures of opsin from native rhodopsin (Park et al. 2008; Scheerer et al.
2008).
In contrast to rhosopsin, GPCRs for hormones and neurotransmitters are not
expressed in tissues at sufficient levels for biophysical studies and are much less
stable when extracted from membranes with detergents. Nevertheless, through
incremental improvements in insect cell expression and the efficiency of the purification
procedure we were able to produce sufficient quantities of β2AR (1–10
milligrams) to start crystallography trials around 1998. However, it wasn’t until
2004 that we obtained the first crystals of the β2AR large enough to examine by
X-ray diffraction. These crystals were still very small (<50 microns) and we were
not able to see diffraction at conventional synchrotron beamlines. I showed a
picture of these crystals to Gebhard Schertler, who was working with Christian
Riekel and Manfred Burghammer at the European Synchrotron Radiation Facility
(ESRF) in Grenoble to develop a microfocus beamline. Gebhard invited me
to bring some of our β2AR crystals to the ESRF. Using a high intensity 5 micron
beam, we were able to see diffraction compatible with a protein crystal at a
resolution of approximately 20Å. While we were disappointed in the poor quality
of the diffraction, we were encouraged by the fact that we were able to form
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200 The Nobel Prizes
crystals of the β2AR.This was an important milestone in the effort and suggested
that a crystal structure of the β2AR was not an impossible goal.
In 2005 Dan Rosenbaum and Søren Rasmussen, two very talented and intrepid
postdoctoral fellows, joined the crystallography effort. Based on our
experience at that time, we speculated that there were two impediments to
crystallography: the dynamic character of the protein, and the very small polar
surface area available for crystal lattice contacts. Our biophysical and biochemical
studies had suggested that the β2AR was a flexible, dynamic protein with
the cytoplasmic ends of TM5 and TM6, and the intervening third intracellular
loop as being the most flexible. We speculated that the dynamic character of
this region of the receptor led to conformational heterogeneity that prevented
the formation of well-ordered crystals. At the same time, biochemical studies
showed that the largest stretches of polar amino acids were largely unstructured
and not suitable for forming crystal lattice contacts. Søren and Dan took
two different approaches to address these problems and to generate better
quality crystals of the β2AR. Søren identified a monoclonal antibody fragment
(Fab) that bound to the cytoplasmic ends of TM5 and TM6. This antibody came
out of a collaboration I initiated in 2003 with Dan Rohr at Medarex, a company
specializing in therapeutic antibodies. The goal of the collaboration was to generate
antibodies that recognized a three-dimensional epitope on native β2AR for
use in crystallography. As an immunogen, I prepared purified β2AR reconstituted
into phospholipid vesicles to maintain its native conformation. We obtained 13
different monoclonal antibodies from Medarex, and Søren and colleagues in the
lab subsequently identified one that bound only to native β2AR and localized its
binding site to a region between TM5 and TM6 (Day et al. 2007).
As an alternative strategy, Dan used protein engineering to replace the same
flexible, dynamic region of the β2AR between TM5 and TM6 of the β2AR with T4
lysozyme (T4L) (Rosenbaum et al. 2007). T4 lysozyme was chosen because it is
a very stable and highly crystallizable soluble protein with amino and carboxyl
termini well positioned to fit between TM5 and TM6.
Both approaches were designed to minimize conformational flexibility, or
at a minimum, mask the most dynamic surface of the receptor and at the same
time increase the amount of polar surface area available for forming crystal lattice
contacts. During 2006 we obtained crystals using both approaches combined
with a lipid-based media known as bicelles (consisting of a mixture of
lipid and detergent) that had been shown to be suitable for membrane protein
crystallization(Faham and Bowie 2002). Initial crystals of the β2AR-Fab and the
β2AR-T4L fusion protein complex both diffracted to below 4 Å. We subsequently
obtained a 3.4Å structure of the β2AR-Fab complex grown in bicelles (Rasmussen
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The Structural Basis of G Protein Coupled Receptor Signaling201
et al. 2007). This was our first look at the three dimensional structure of the β2AR,
but a higher resolution structure would soon follow (Fig. 1A).
In the fall of 2006 we sent purified β2AR-T4L complex to Vadim Cherezov in
the lab of Raymond Stevens at Scripps. Vadim had trained with Martin Caffrey
at the Ohio State University. Martin’s lab had recently developed miniaturized,
high-throughput methods for lipidic cubic phase (LCP) crystallography (Cheng
et al. 1998; Misquitta et al. 2004). We previously explored the use of LCP methods
to crystallize the β2AR in 1999 in collaboration with Peter Nollert; however,
at that time the methods were very labor intensive and used relatively large
amounts of protein to screen very few conditions. The methods developed
in Martin’s lab together with the robot built by his team enabled screening of
thousands of conditions with a few milligrams of protein (Cherezov et al. 2004).
Vadim had recently joined the Stevens lab, bringing with him an LCP robot on
loan from Martin Caffrey. This collaboration led to a 2.4 Å structure of the β2ART4L
complex (Cherezov et al. 2007; Rosenbaum et al. 2007) (Fig.2B). The fusion
protein strategy developed for the β2AR has since been successfully applied
to a growing number of other GPCRs. Through collaborative efforts with several
other groups, my lab recently used the same fusion protein approach to
Figure 2.  First crystal structures of β2AR in the inactive states. A. The β2AR-Fab complex.
B. The β2AR-T4 Lysozyme (T4L) fusion protein.
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202 The Nobel Prizes
determine structures of the M2 and M3 muscarinic receptors (Haga et al. 2012;
Kruse et al. 2012), the mu and delta opioid receptors (Granier et al. 2012; Manglik
et al. 2012), and the protease activated receptor PAR1(Chung et al. 2013). More
recently we have found that fusing T4L to the amino terminus of the β2AR and
simply deleting most of the third intracellular loop can also facilitate crystallization
(Zou et al. 2012).
Another approach that has succeeded in obtaining GPCR crystal structures
involves scanning mutagenesis to identify thermostabilizing mutations. Chris
Tate and Gebhard Schertler and their colleagues pioneered this approach to
obtain the structure of the avian β1AR (Warne et al. 2008). These stabilizing mutations
may reduce structural flexibility and permit the use of detergents having
a smaller micelle size. This approach has also been used to obtain the structure
of the adenosine A2A receptor (Lebon et al. 2011) and, in combination with a T4
lysozyme fusion protein strategy, the neurotensin receptor (White et al. 2012).
Capturing active states by crystallography
Immediately after obtaining these initial inactive-state structures of the β2AR we
initiated efforts to capture actives states by crystallography. Using the methods
that were successful in obtaining inactive-state structures, we were not able to
obtain crystals of a β2AR bound to an agonist. Our concern was that due to
the relatively low affinity of agonists (when compared to the very high affinity
inverse-agonist carazolol used to obtain inactive-state structures), we had
incomplete occupancy of the receptor under crystallography conditions. This
would lead to conformational heterogeneity. To overcome this problem, Dan
Rosenbaum worked with Ralph Holl and Peter Gmeiner (University of Erlangen)
to develop a covalent agonist for the β2AR. Using this approach they were able
to obtain crystals of the covalent agonist bound β2AR; however, the cytoplasmic
face of the receptor was indistinguishable from the inactive-state structure
(Rosenbaum et al. 2011).
These disappointing results were consistent with what we had learned from
earlier biophysical studies, that agonists alone do not fully stabilize the active
state of the β2AR. This was first observed using fluorescence spectroscopy (Ghanouni
et al. 2001; Yao et al. 2009) and confirmed in more recent studies using
NMR spectroscopy (Nygaard et al. 2013). Figure 3 is a cartoon illustrating the
dynamic character of the receptor, showing that the receptor exists as an ensemble
of conformations. Due to the flexibility of the unliganded β2AR, a small
population can be in an active conformation, accounting for the phenomenon
of basal activity. On binding the agonist, the cytoplasmic interface becomes
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The Structural Basis of G Protein Coupled Receptor Signaling203
even more dynamic, sampling a broader spectrum of conformations. This contributes
to the challenges in crystallizing agonist-bound receptor. Fluorescence
and NMR experiments showed us that stabilization of the active state required
that the receptor must form a complex with its G protein, or some other protein
that binds to and stabilizes the active conformation (Yao et al. 2009; Nygaard et
al. 2013).
Our efforts to crystallize the β2AR-Gs complex were in progress, but not
yet successful. As an alternative we were exploring antibodies and other binding
proteins. In May of 2009 I had the good fortune to meet Jan Steyaert (Free
University of Brussels) at a Gordon Conference in Italy. Jan was pioneering the
application of single-chain camelid antibody fragments, known as nanobodies,
as facilitators of protein crystallogenesis. Shortly after the conference I sent Jan
purified, agonist-bound β2AR reconstituted into phospholipid vesicles for immunizing
llamas. By November 2009 we had our first nanobodies and Søren
Rasmussen identified one that exhibited G protein-like properties. This nanobody
(Nb80) bound to purified β2AR and allosterically enhanced agonist binding
affinity by 100-fold, similar to what is observed in a β2AR-Gs complex. This
β2AR-Nb80 complex gave us the first picture of the active-state conformation of
the β2AR (Rasmussen et al. 2011) (Fig. 4A).
Figure 3.  Cartoon illustrating the dynamic character of the β2AR. In the absence of
a ligand, the G protein coupling interface of the receptor exists in an ensemble of
predominantly low energy conformations. Rare active-state conformations are responsible
for basal activity. Agonist binding increases the dynamic properties of the β2AR,
increasing the probably of active-state conformations. Only G protein binding can fully
stabilize the active state.
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204 The Nobel Prizes
The β2AR-Gs Complex
In 2005 I met Roger Sunahara (University of Michigan) at a Gordon Conference
and we began working together to understand the structural basis of cooperative
interactions between the β2AR and its G protein Gs. Our long-term goal
was to crystallize the β2AR-Gs complex. The ultimate success of this effort would
require an extensive network of collaborations with investigators from diverse
disciplines.
One of the most important contributions to this effort was the use of single
particle electron microscopy (EM) to provide structural insights that guided our
crystallization strategy. By 2009 Søren Rasmussen in my lab and Brian Devree in
Roger’s lab were making considerable progress on the biochemistry of the complex.
They were able to form a relatively stable β2AR-Gs complex that migrated
as a single peak by size exclusion chromatography; however we were not able
to grow crystals. We sent protein to Georgios Skiniotis, an expert in single particle
EM methods at the University of Michigan. Our first view of the β2AR-Gs complex
came from a low-resolution structure generated from negative stained EM
images. This structure revealed an unexpected feature of the complex that was
one of the biggest obstacles to crystal growth. The Gαs subunit is composed
of an alpha helical domain and a Ras-like domain with the GCP binding pocket
at the interface. The EM structure revealed that the alpha helical domain of the
Figure 4.  Active-state structures of the human β2AR.
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The Structural Basis of G Protein Coupled Receptor Signaling205
Gs alpha subunit was conformationally heterogeneous. Subsequent EM studies
helped to identify chemical additives that minimized the conformational heterogeneity,
as well as a nanobody (Nb35) that stabilized the complex.
Other contributions to the success of the β2AR-Gs crystallogrphy include the
identification of an ultra high affinity agonist (BI-167107) from Boehringer Ingelheim.
This agonist has a dissociation half-life of more than 30 hours, ensuring
that the β2AR would be occupied by an agonist at all times. Another important
reagent was a new detergent, MNG-3, provided by Pil Seok Chae and Sam Gellman
at the University of Wisconsin, Madision (Chae et al. 2010). This detergent
stabilized the β2AR-Gs complex during incorporation into the mesophase lipid
used for crystallography. Martin Caffrey provided a special mesophase lipid (7.7
MAG) that enabled the application of lipidic cubic phase methods to a large
protein complex (Misquitta et al. 2004).To further stabilize the β2AR-Gs complex,
we worked with Jan Steyaert to develop a nanobody (Nb35) that bound to the
interface between the alpha and beta subunits of Gs. Finally, replacement of the
amino terminus of the β2AR with T4 lysozyme facilitated packing interactions
with the extracellular surface.
The first crystals of the β2AR-Gs complex were obtained in April 2011 and
we were ultimately able to solve the structure to 3.2Å (Fig. 4B) (Rasmussen et
al. 2011). The structure revealed how the binding of a small agonist at the extracellular
side of the receptor propagates structural changes across the lipid
bilayer to effect activation of a cytosolic G protein. In this process, small structural
changes around the binding pocket are amplified to very large structural
changes in the G protein.
Figures 5–8 follow the process of activation from agonist-stabilized changes
in the β2AR to receptor mediated changes in Gs. As shown in Figure 5, structural
differences between the inactive and active state β2AR structures are relatively
small, particularly around the ligand binding pocket.The largest changes are observed
at the cytoplasmic surface including a 14Å outward movement of TM6.
Fig. 6 compares the ligand binding pockets for active and inactive structures.
Amino acids within 4Å of the agonist BI167107 are shown. Changes in the
binding pocket are relatively subtle, with the agonist pocket being smaller than
that of the inverse agonist bound receptor. The largest difference is a 2Å inward
movement around Ser207 in TM5. Ser203, 204 and 207 have previously been
shown to be important for agonist binding and activation.
To understand how these small changes in the binding pocket contribute
to the larger 14Å movement at the cytoplasmic end of the receptor, we
looked for the changes in packing interactions between TM segments below
the ligand binding pocket. As shown in Figure 7, a set of conserved amino acids
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206 The Nobel Prizes
Figure 5.  A comparison of the carazolol-bound, inactive-state structure of the β2AR
(gray) and the active-state structure of the β2AR (green) from the β2AR-Gs complex.
Figure 6.  A comparison of the binding pocket for the inverse agonist carazolol in the
inactive-state structure of the β2AR (gray) and the BI167107 binding pocket for the
active-state structure of the β2AR (green).
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The Structural Basis of G Protein Coupled Receptor Signaling207
Figure 7.  Packing interactions of conserved amino acids in inactive and active states
of the β2AR. Only TM3, TM5, TM6 and TM7 are shown. A. A set of conserved amino acids
(represented as spheres) pack together to stabilize the inactive conformation (Fig. 7A).
These include the highly conserved Pro211 in TM5 and Phe282 in TM6 as well as Ile121
in TM3 and Asn318 in TM7. B. Active state. The packing arrangement observed in the
inactive state is not compatible with the small inward movement of TM5 and requires a
rearrangement to accommodate the agonist bound active state. C. Active and inactive
structures are superimposed. Pro 211, Phe282, Ile121 and Asn318 are represented as
sticks to more easily compare differences in position.
pack together to stabilize the inactive conformation (Fig. 7A). These include the
highly conserved Pro 211 in TM5 and Phe 282 in TM6 as well as Ile 121 in TM3
and Asn 318 in TM7. This packing arrangement is not compatible with the small
inward movement of TM5 and requires a rearrangement to accommodate the
agonist bound active state (Fig. 7B). This rearrangement involves a rotation of
TM6 around Phe282 that is largely responsible for the large outward movement
at its cytoplasmic end (Fig. 7C).
Figure 8 shows the structural changes in Gs upon forming a complex with
agonist-bound β2AR. The inactive state of the Gs heterotrimer is modeled from
the crystal structure of the Gi heterotrimer. The Gαs subunit is composed of a
Ras-like GTPase domain and an α-helical domain.The GDP binding site occupies
the interface between these two domains. On coupling to the β2AR. the Gαs
subunit undergoes large structural changes, with the α-helical domain being
displaced by approximately 130º. The carboxyl terminal α5-helix of the Ras-like
domain is displaced 5Å into the core of the active receptor, stabilizing the active
state of the receptor. This displacement of the α5-helix as well as more subtle
changes transmitted from the receptor to the GDP binding pocket through the
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208 The Nobel Prizes
β1-strand of the Ras domain are responsible for dissociation of GDP. The emptystate
β2AR-Gs complex is poised for activation by GTP. The very large displacement
of alpha helical domain was not expected from prior studies; however,
as noted above, low-resolution single particle EM studies by Georgios Skiniotis
revealed that the alpha helical domain is highly dynamic.
Conclusions
The β2AR–Gs complex crystal structure provides the first high-resolution view
of transmembrane signaling for a GPCR. We now have a framework to design
experiments to investigate the mechanism of complex formation, GTP binding
and complex dissociation. Of particular interest will be studies designed to determine
the functional significance of the large movement of the Gαs α-helical
domain relative to the Ras-like domain that is observed in the β2AR–Gs complex.
Nevertheless, the β2AR-Gs structure leaves an important question unanswered.
It does not explain why the β2AR preferentially couples to Gs. While some of the
β2AR sequences involved in the β2AR-Gs interface have been shown to have a
role in G protein coupling; there is no clear consensus sequence for Gs-coupling
specificity when these segments are aligned with other Gs-coupled GPCRs.
Coupling specificity may be dictated by interactions between the β2AR and
Gs that precede the formation of the nucleotide-free complex. While the studies
outlined in this lecture have advanced the field, much work remains to be
done before we can fully understand and pharmacologically control signaling
by these fascinating membrane proteins.
Figure 8.  Comparison of inactive GDP bound Gs (left panel) with the β2AR-Gs complex
(middle panel). The two structures are superimposed in the right panel. The GDP-bound
Gs heterotrimer was modeled from the crystal structure of the Gi heterotrimer.
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The Structural Basis of G Protein Coupled Receptor Signaling209
ACKNOWLEDGEMENTS
This lecture represents work done since 1984 and reflects contributions of many
colleagues, postdoctoral fellows and graduate students, as well as inspiration
provided by the work of other investigators in the field. I want to single out a
few colleagues for special thanks. My wife Tong Sun Kobilka has been my colleague,
advisor and strongest advocate for over thirty years. We began working
together at Duke, and continue to this day. I want to thank Bob Lefkowitz for his
mentorship during my fellowship and beyond. Bill Weis has been my colleague
and advisor on all matters having to do with interpreting diffraction data and
solving difficult crystal structures. I want to thank Roger Sunahara for initiating
our very enjoyable and fruitful collaboration on the β2AR-Gs structure. Finally,
I want to thank the other members of the team of scientists that contributed
to the β2AR-Gs structural effort. At Stanford: Søren Rasmussen, Foon Sun Thian,
Yaozhong Zou, Andrew Kruse, Ka Young Chung, and Jesper Mathiesen. At the
University of Michigan: Brian DeVree, Diane Calinski, GerwinWestfield, and Georgios
Skiniotis. At the University of Wisconsin, Madison: Pil Seok Chae and Sam
Gellman. At the Free University of Brussels: Els Pardon and Jan Steyaert. At Trinity
College Dublin: Joseph Lyons, Syed Shah and Martin Caffrey. This work has been
supported by grants from the National Institutes of Health (NINDS and NIGMS),
and gifts from the Mathers Foundation and Lundbeck.
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small viscous lipid-containing samples,”Chem Phys Lipids 95(1): 11–21.
4.	 Cherezov, V., A. Peddi, L. Muthusubramaniam,Y. F. Zheng and M. Caffrey (2004),“A robotic
system for crystallizing membrane and soluble proteins in lipidic mesophases,”
Acta Crystallogr D Biol Crystallogr 60(Pt 10): 1795–1807.
5.	 Cherezov,V., D. M. Rosenbaum, M. A. Hanson, S. G. Rasmussen, F. S.Thian,T. S. Kobilka,
H. J. Choi, P. Kuhn, W. I. Weis, B. K. Kobilka and R. C. Stevens (2007), “High-resolution
crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor,”Science
318(5854): 1258–1265.
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210 The Nobel Prizes
6.	 Chung, K.Y., P.W. Day, G.Velez-Ruiz, R. K. Sunahara and B. K. Kobilka (2013),“Identification
of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based
Proteomic Approach,”PLoS One 8(1): e54942.
7.	 Day, P. W., S. G. Rasmussen, C. Parnot, J. J. Fung, A. Masood, T. S. Kobilka, X. J. Yao, H.
J. Choi, W. I. Weis, D. K. Rohrer and B. K. Kobilka (2007),“A monoclonal antibody for G
protein-coupled receptor crystallography,”Nat Methods 4(11): 927–929.
8.	 Deupi, X. and B. Kobilka (2007),“Activation of G protein-coupled receptors,”Adv Protein
Chem 74: 137–166.
9.	 Deupi, X. and B. K. Kobilka (2010), “Energy landscapes as a tool to integrate GPCR
structure, dynamics, and function,”Physiology (Bethesda) 25(5): 293–303.
10.	 Dixon, R. A., B. K. Kobilka, D. J. Strader, J. L. Benovic, H. G. Dohlman, T. Frielle, M. A.
Bolanowski, C. D. Bennett, E. Rands, R. E. Diehl, R. A. Mumford, E. E. Slater, I. S. Sigal,
M. G. Caron, R. J. Lefkowitz and C. D. Strader (1986),“Cloning of the gene and cDNA
for mammalian beta-adrenergic receptor and homology with rhodopsin,” Nature
321(6065): 75–79.
11.	 Faham, S. and J. U. Bowie (2002),“Bicelle crystallization: a new method for crystallizing
membrane proteins yields a monomeric bacteriorhodopsin structure,”J Mol Biol
316(1): 1–6.
12.	 Gether, U., J. A. Ballesteros, R. Seifert, E. Sanders-Bush, H. Weinstein and B. K. Kobilka
(1997), “Structural instability of a constitutively active G protein-coupled receptor.
Agonist-independent activation due to conformational flexibility,” J Biol Chem
272(5): 2587–2590.
13.	 Gether, U., S. Lin, P. Ghanouni, J. A. Ballesteros, H. Weinstein and B. K. Kobilka (1997),
“Agonists induce conformational changes in transmembrane domains III and VI of
the beta2 adrenoceptor,”Embo J 16(22): 6737–6747.
14.	 Gether, U., S. Lin and B. K. Kobilka (1995),“Fluorescent labeling of purified beta 2 adrenergic
receptor. Evidence for ligand-specific conformational changes,”J Biol Chem
270(47): 28268–28275.
15.	 Ghanouni, P., Z. Gryczynski, J. J. Steenhuis, T. W. Lee, D. L. Farrens, J. R. Lakowicz and
B. K. Kobilka (2001), “Functionally different agonists induce distinct conformations
in the G protein coupling domain of the beta 2 adrenergic receptor,” J Biol Chem
276(27): 24433–24436.
16.	 Granier, S., A. Manglik, A. C. Kruse, T. S. Kobilka, F. S. Thian, W. I. Weis and B. K. Kobilka
(2012), “Structure of the delta-opioid receptor bound to naltrindole,” Nature
485(7398): 400–404.
17.	 Guan, X. M., T. S. Kobilka and B. K. Kobilka (1992),“Enhancement of membrane insertion
and function in a type IIIb membrane protein following introduction of a cleavable
signal peptide,”J Biol Chem 267(31): 21995–21998.
18.	 Guan, X. M., S. J. Peroutka and B. K. Kobilka (1992), “Identification of a single amino
acid residue responsible for the binding of a class of beta-adrenergic receptor antagonists
to 5-hydroxytryptamine1A receptors,”Mol Pharmacol 41(4): 695–698.
19.	 Haga, K., A. C. Kruse, H. Asada, T. Yurugi-Kobayashi, M. Shiroishi, C. Zhang, W. I. Weis,
T. Okada, B. K. Kobilka, T. Haga and T. Kobayashi (2012), “Structure of the human
M2 muscarinic acetylcholine receptor bound to an antagonist,” Nature 482(7386):
547–551.
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The Structural Basis of G Protein Coupled Receptor Signaling211
20.	 Kobilka, B. K. (1990), “The role of cytosolic and membrane factors in processing of
the human beta-2 adrenergic receptor following translocation and glycosylation in
a cell-free system,”J Biol Chem 265(13): 7610–7618.
21.	 Kobilka, B. K. (1995),“Amino and carboxyl terminal modifications to facilitate the production
and purification of a G protein-coupled receptor,” Analyt Biochem 231(1):
269–271.
22.	 Kobilka, B. K., T. S. Kobilka, K. Daniel, J. W. Regan, M. G. Caron and R. J. Lefkowitz
(1988), “Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains
involved in effector coupling and ligand binding specificity,” Science 240(4857):
1310–1316.
23.	 Kruse, A. C., J. Hu, A. C. Pan, D. H. Arlow, D. M. Rosenbaum, E. Rosemond, H. F. Green,T.
Liu, P. S. Chae, R. O. Dror, D. E. Shaw, W. I. Weis, J. Wess and B. K. Kobilka (2012),“Structure
and dynamics of the M3 muscarinic acetylcholine receptor,” Nature 482(7386):
552–556.
24.	 Lebon, G., T. Warne, P. C. Edwards, K. Bennett, C. J. Langmead, A. G. Leslie and C. G.
Tate (2011),“Agonist-bound adenosine A2A receptor structures reveal common features
of GPCR activation,”Nature 474(7352): 521–525.
25.	 Lin, S., U. Gether and B. K. Kobilka (1996),“Ligand stabilization of the beta 2 adrenergic
receptor: effect of DTT on receptor conformation monitored by circular dichroism
and fluorescence spectroscopy,”Biochemistry 35(46): 14445–14451.
26.	 Manglik, A., A. C. Kruse, T. S. Kobilka, F. S. Thian, J. M. Mathiesen, R. K. Sunahara, L.
Pardo, W. I. Weis, B. K. Kobilka and S. Granier (2012), “Crystal structure of the muopioid
receptor bound to a morphinan antagonist,”Nature 485(7398): 321–326.
27.	 Misquitta, L. V., Y. Misquitta, V. Cherezov, O. Slattery, J. M. Mohan, D. Hart, M. Zhalnina,
W. A. Cramer and M. Caffrey (2004),“Membrane protein crystallization in lipidic mesophases
with tailored bilayers,”Structure 12(12): 2113–2124.
28.	 Mizobe, T., M. Maze, V. Lam, S. Suryanarayana and B. K. Kobilka (1996),“Arrangement
of transmembrane domains in adrenergic receptors. Similarity to bacteriorhodopsin,”J
Biol Chem 271(5): 2387–2389.
29.	 Nygaard, R.,Y. Zou, R. O. Dror,T. J. Mildorf, D. H. Arlow, A. Manglik, A. C. Pan, C. W. Liu, J.
J. Fung, M. P. Bokoch, F. S. Thian, T. S. Kobilka, D. E. Shaw, L. Mueller, R. S. Prosser and B.
K. Kobilka (2013), “The Dynamic Process of beta(2)-Adrenergic Receptor Activation,”
Cell 152(3): 532–542.
30.	 Okada, T., I. Le Trong, B. A. Fox, C. A. Behnke, R. E. Stenkamp and K. Palczewski (2000),
“X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained
from mixed micelles,”J Struct Biol 130(1): 73–80.
31.	 Palczewski, K., T. Kumasaka, T. Hori, C. A. Behnke, H. Motoshima, B. A. Fox, I. Le Trong,
D. C. Teller, T. Okada, R. E. Stenkamp, M. Yamamoto and M. Miyano (2000), “Crystal
structure of rhodopsin: A G protein-coupled receptor,”Science 289(5480): 739–745.
32.	 Park, J. H., P. Scheerer, K. P. Hofmann, H. W. Choe and O. P. Ernst (2008), “Crystal
structure of the ligand-free G-protein-coupled receptor opsin,” Nature 454(7201):
183–187.
33.	 Rasmussen, S. G., H. J. Choi, J. J. Fung, E. Pardon, P. Casarosa, P. S. Chae, B. T. Devree,
D. M. Rosenbaum, F. S. Thian, T. S. Kobilka, A. Schnapp, I. Konetzki, R. K. Sunahara, S.
H. Gellman, A. Pautsch, J. Steyaert, W. I. Weis and B. K. Kobilka (2011), “Structure of
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212 The Nobel Prizes
a nanobody-stabilized active state of the beta(2) adrenoceptor,” Nature 469(7329):
175–180.
34.	 Rasmussen, S. G., H. J. Choi, D. M. Rosenbaum, T. S. Kobilka, F. S. Thian, P. C. Edwards,
M. Burghammer, V. R. Ratnala, R. Sanishvili, R. F. Fischetti, G. F. Schertler, W. I. Weis
and B. K. Kobilka (2007),“Crystal structure of the human beta2 adrenergic G-proteincoupled
receptor,”Nature 450(7168): 383–387.
35.	 Rasmussen, S. G., B. T. DeVree, Y. Zou, A. C. Kruse, K. Y. Chung, T. S. Kobilka, F. S. Thian,
P. S. Chae, E. Pardon, D. Calinski, J. M. Mathiesen, S. T. Shah, J. A. Lyons, M. Caffrey, S.
H. Gellman, J. Steyaert, G. Skiniotis, W. I. Weis, R. K. Sunahara and B. K. Kobilka (2011),
“Crystal structure of the beta2 adrenergic receptor-Gs protein complex,” Nature
477(7366): 549–555.
36.	 Rosenbaum, D. M.,V. Cherezov, M. A. Hanson, S. G. Rasmussen, F. S.Thian,T. S. Kobilka,
H. J. Choi, X. J.Yao, W. I. Weis, R. C. Stevens and B. K. Kobilka (2007),“GPCR engineering
yields high-resolution structural insights into beta2-adrenergic receptor function,”
Science 318(5854): 1266–1273.
37.	 Rosenbaum, D. M., C. Zhang, J. A. Lyons, R. Holl, D. Aragao, D. H. Arlow, S. G. Rasmussen,
H. J. Choi, B. T. Devree, R. K. Sunahara, P. S. Chae, S. H. Gellman, R. O. Dror, D. E.
Shaw, W. I. Weis, M. Caffrey, P. Gmeiner and B. K. Kobilka (2011), “Structure and function
of an irreversible agonist-beta(2) adrenoceptor complex,” Nature 469(7329):
236–240.
38.	 Scheerer, P., J. H. Park, P. W. Hildebrand, Y. J. Kim, N. Krauss, H. W. Choe, K. P. Hofmann
and O. P. Ernst (2008), “Crystal structure of opsin in its G-protein-interacting conformation,”Nature
455(7212): 497–502.
39.	 Shenoy, S. K., M. T. Drake, C. D. Nelson, D. A. Houtz, K. Xiao, S. Madabushi, E. Reiter, R.
T. Premont, O. Lichtarge and R. J. Lefkowitz (2006),“Beta-arrestin-dependent, G protein-independent
ERK1/2 activation by the beta2 adrenergic receptor,” J Biol Chem
281(2): 1261–1273.
40.	 Suryanarayana, S., D. A. Daunt, M. Von Zastrow and B. K. Kobilka (1991), “A point
mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor
increases its affinity for a family of beta receptor antagonists,” J Biol Chem 266(23):
15488–15492.
41.	 Suryanarayana, S. and B. K. Kobilka (1993),“Amino acid substitutions at position 312
in the seventh hydrophobic segment of the beta 2-adrenergic receptor modify
ligand-binding specificity,”Mol Pharmacol 44(1): 111–114.
42.	 Swaminath, G., X. Deupi, T. W. Lee, W. Zhu, F. S. Thian, T. S. Kobilka and B. Kobilka
(2005), “Probing the beta2 Adrenoceptor Binding Site with Catechol Reveals Differences
in Binding and Activation by Agonists and Partial Agonists,” J Biol Chem
280(23): 22165–22171.
43.	 Swaminath, G., Y. Xiang, T. W. Lee, J. Steenhuis, C. Parnot and B. K. Kobilka (2004),
“Sequential Binding of Agonists to the {beta}2 Adrenoceptor: Kinetic evidence for
intermediate conformational states,”J Biol Chem 279(1): 686–691.
44.	 Terrillon, S. and M. Bouvier (2004), “Roles of G-protein-coupled receptor dimerization,”EMBO
Rep 5(1): 30–34.
45.	 Unger, V. M., P. A. Hargrave, J. M. Baldwin and G. F. Schertler (1997), “Arrangement of
rhodopsin transmembrane alpha-helices,”Nature 389(6647): 203–206.
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The Structural Basis of G Protein Coupled Receptor Signaling213
46.	 Warne, T., M. J. Serrano-Vega, J. G. Baker, R. Moukhametzianov, P. C. Edwards, R. Henderson,
A. G. Leslie, C. G. Tate and G. F. Schertler (2008), “Structure of a beta1-adrenergic
G-protein-coupled receptor,”Nature 454(7203): 486–491.
47.	 White, J. F., N. Noinaj,Y. Shibata, J. Love, B. Kloss, F. Xu, J. Gvozdenovic-Jeremic, P. Shah,
J. Shiloach, C. G. Tate and R. Grisshammer (2012), “Structure of the agonist-bound
neurotensin receptor,”Nature 490(7421): 508–513.
48.	 Wisler, J. W., S. M. DeWire, E. J. Whalen, J. D. Violin, M. T. Drake, S. Ahn, S. K. Shenoy
and R. J. Lefkowitz (2007), “A unique mechanism of beta-blocker action: Carvedilol
stimulates beta-arrestin signaling,”Proceedings of the National Academy of Sciences of
the United States of America 104(42): 16657–16662.
49.	 Xiao, R. P., P. Avdonin,Y.Y. Zhou, H. Cheng, S. A. Akhter,T. Eschenhagen, R. J. Lefkowitz,
W. J. Koch and E. G. Lakatta (1999), “Coupling of beta2-adrenoceptor to Gi proteins
and its physiological relevance in murine cardiac myocytes,”Circ Res 84(1): 43–52.
50.	 Yao, X. J., G. Velez Ruiz, M. R. Whorton, S. G. Rasmussen, B. T. DeVree, X. Deupi, R. K.
Sunahara and B. Kobilka (2009), “The effect of ligand efficacy on the formation and
stability of a GPCR-G protein complex,”Proc Natl Acad Sci U S A 106(23): 9501–9506.
51.	 Zou, Y., W. I. Weis and B. K. Kobilka (2012), “N-terminal T4 lysozyme fusion facilitates
crystallization of a G protein coupled receptor,”PLoS One 7(10): e46039.
Portrait photo of professor Kobilka by photographer Ulla Montan.
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