MINIREVIEW
G Protein-coupled Receptors
II. MECHANISM OF AGONIST
ACTIVATION*
Ulrik
Gether
and
Brian K.
Kobilka§¶
From the
Department of Cellular Physiology, Institute
of Medical Physiology 12.5, The Panum Institute, University of
Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and
§ Howard Hughes Medical Institute and Division of
Cardiovascular Medicine, Stanford University Medical School,
Stanford, California 94305
 |
INTRODUCTION |
The majority of transmembrane signal
transduction in response to hormones and neurotransmitters is mediated
by G protein-coupled receptors
(GPCRs).1 Moreover, GPCRs are
the principal signal transducers for the senses of sight and smell.
GPCRs are characterized by seven membrane-spanning domains with an
extracellular N terminus and a cytoplasmic C terminus (Fig.
1) (GPCR structure reviewed in Refs.
1-3). Based on certain key sequences, GPCRs can be divided into three
major subfamilies, receptors related to rhodopsin (type A), receptors
related to the calcitonin receptor (type B), and receptors related to
the metabotropic receptors (type C). Of these, the rhodopsin subfamily is by far the largest and most extensively investigated and will be the
focus of the present review.

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Fig. 1.
Two-dimensional model of the 2
receptor illustrating the key structural features of a GPCR belonging
to the "rhodopsin-like" subfamily. The most conserved residue
in each transmembrane segments is indicated both using the Schwartz
nomenclature (59) and the Ballesteros-Weinstein nomenclature (15). In
the Schwartz nomenclature the most conserved residue in each helix had
been given a generic number according to its position in the helix. In
the Ballesteros-Weinstein nomenclature the most conserved residue in
each helix had been given the number 50. A series of conserved
tryptophans and prolines are indicated in blue. The kink
that may be caused by ProVI:15 has been suggested to be critical for
the conformational changes involved in receptor activation (49). The
almost invariable disulfide bridge between extracellular loops 2 and 3 and the conserved palmitoylation site in the C-terminal tail are
indicated in white. Residues AspIII:08, SerV:12, SerV:16,
PheVI:17, and AsnVI:20 shown to be involved in binding of epinephrine
to the 2 adrenergic receptor are indicated in
red (3, 60). Mutations at Ala293
(green) in the 1b receptor lead to
agonist-independent activation (31). Other residues are discussed in
the text.
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GPCR domains involved in ligand binding are nearly as diverse as the
chemical structures of the known agonists (1, 3, 5). Small molecular
weight ligands bind to sites within the hydrophobic core formed by the
transmembrane (TM)
-helices, whereas binding sites for peptides and
protein agonists include the N terminus and extracellular hydrophilic
loops joining the transmembrane domains (2, 5). Domains critical for
interaction with the G protein have been localized to the second and
third cytoplasmic loops and the C terminus (2).
High resolution structural analysis of GPCRs has been hindered by their
low natural abundance and the difficulty in producing and purifying
significant quantities of recombinant protein. A low resolution
structure of rhodopsin obtained from electron diffraction of
two-dimensional crystals has been useful in predicting the arrangement
and relative orientation of the transmembrane domains (6, 7) (Fig.
2A). Mutagenesis studies aimed
at identifying intramolecular interactions have provided evidence for a
similar arrangement in other GPCRs (8-12), and several molecular
models of different GPCRs have been developed (13-15).

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Fig. 2.
A, arrangement of
transmembrane domains of a prototypical G protein-coupled receptor as
viewed from the extracellular surface of the membrane (based on the
projection maps from two-dimensional crystals of rhodopsin (7)).
B, proposed conformational changes in TM3 and TM6 following
agonist activation based on studies of rhodopsin and the
2 adrenergic receptor.
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This review will examine current progress in addressing a fundamental
question in the field of GPCR-mediated signal transduction: the nature
of the physical changes in receptors that link agonist binding to G
protein activation. It will explore the differences and similarities in
the activation of rhodopsin and other GPCRs by focusing on two
questions. What conformational changes take place during receptor
activation? How do agonists induce these conformational changes in the
receptor?
 |
Extended Ternary Complex Model |
Perhaps the most widely accepted model used to describe agonist
activation of GPCRs is the ternary complex model, which accounts for
the cooperative interactions among receptor, G protein, and agonist
(16). This model has recently been extended to accommodate the
observation that many receptors can activate G proteins in the absence
of agonist and that mutations in different structural domains of the
receptor can enhance this agonist-independent activity (17, 18). The
extended ternary complex model also accounts for the effects of
different classes of drugs (full agonists, partial agonists, neutral
antagonists, and inverse agonists) on receptor signaling. The model
proposes that the receptor exists in an equilibrium of two functionally
distinct states: the inactive (R) and the active
(R*) state. In the absence of agonists, the level of basal
receptor activity is determined by the equilibrium between R
and R*. The efficacy of ligands is thought to be a
reflection of their ability to alter the equilibrium between these two
states. Whereas most properties of GPCRs can be explained by this
model, several studies suggest that a more complex model may be
necessary (reviewed by Kenakin (19, 20)). Nevertheless, for the purpose
of our discussion we will use the terms R to refer to the
inactive conformation of the receptor and R* to refer to the
conformation capable of activating G proteins.
 |
Agonist-induced Conformational Changes |
Is Dimerization Involved in Receptor
Activation?--
Agonist-induced receptor dimerization is required for
signal transduction for several non-GPCR receptor families having
single membrane-spanning domains (such as receptor tyrosine kinases and the growth hormone receptor family (21)). Several recent reports provide evidence that GPCRs can also form dimers, raising the possibility that dimerization is part of the activation process (22-24). However, different mechanisms of dimer formation were observed for different receptors (
2 adrenergic receptor
(22), the
-opioid receptor (23), and the metabotropic glutamate
receptor (24)) suggesting that receptor dimerization is not essential for G protein activation but may play a role in other receptor functions such as subtype-specific receptor regulation.
Evidence for Protonation as a Key Element in Receptor
Activation--
The conserved DRY motif at the cytoplasmic side of TM3
(Fig. 1, orange) is highly conserved in members of the
rhodopsin GPCR family (25). The invariably conserved arginine
(ArgIII:26) in this motif has been hypothesized to be constrained in a
hydrophilic pocket formed by conserved polar residues in TM1, TM2, and
TM7 (Fig. 1, yellow) (14, 26). It has been proposed that
receptor activation involves protonation of AspIII:25 causing
ArgIII:26 to shift out of the polar pocket leading to cytoplasmic
exposure of buried sequences in the second and third intracellular
loops. The hypothesis is supported both by computational simulations and by the observation that mutating AspIII:25 results in increased agonist-independent receptor activity of both the
1b
adrenergic receptor (14, 26) and the
2 adrenergic
receptor.2 Similarly,
mutation of the corresponding GluIII:25 in rhodopsin also causes
constitutive receptor activation (27, 28), and it has been demonstrated
that proton uptake by GluIII:25 in rhodopsin is an important event in
the formation of the activated metarhodopsin II intermediate (29).
GPCRs Are Maintained in an Inactive Conformation by Stabilizing
Intramolecular Interactions--
In several receptors it has been
demonstrated that discrete mutations in the C-terminal region of the
third intracellular loop can cause dramatic increases in
agonist-independent receptor activity (reviewed in Ref. 30).
Replacement of Ala293 in the
1b receptor
(Fig. 1, green) with any other residue resulted in higher
constitutive activity (31). It was therefore proposed that important
conformational constraints maintain the receptor preferentially in an
inactive conformation and that these constraint(s) are released upon
activation (or by specific mutations) causing key sequences to be
exposed to the G protein (30, 31). Recently, this hypothesis has been
supported by the observation that a mutation causing constitutive
activation of the
2 receptor is associated with a marked
structural instability and enhanced conformational flexibility
(32).
There is compelling experimental evidence from several different
receptors supporting the existence of a stabilizing network of
intramolecular interactions constraining the receptor in the inactive
conformation (14, 33-36). In rhodopsin for example, disruption of a
presumed salt bridge between TM3 and TM7 results in constitutive
activation of opsin in the absence of chromophore (33). Experiments on
a series of chimeric luteinizing hormone/follicle-stimulating hormone
receptors provide evidence that important stabilizing interactions
between TM5 and TM6 are responsible for the resistance of the
follicle-stimulating hormone receptor to constitutively activating
mutations observed in the highly homologous luteinizing hormone
receptor (35).
Javitch and co-workers (37) obtained evidence for a conformational
rearrangement of TM6 in a constitutively activated
2 adrenergic receptor, providing additional support for an important role
of TM6 in the network of conformational constraints required to
maintain the receptor in the inactive state. A cysteine in TM6, which
is not accessible to modification by a charged hydrophilic sulfhydryl-specific reagent in the wild type
2 receptor,
becomes accessible in the constitutively active mutant (37). Further support for the importance of the orientation of TM6 relative to TM5
and TM3 in receptor activation comes from studies using engineered
metal ion binding sites. Histidine substitutions can be used to
generate a zinc-binding site between two TM domains. Zinc binding to
engineered metal ion binding sites linking TM6 and TM5 in the NK-1
(substance P) (38) and TM6 and TM3 in rhodopsin (39) prevented receptor
activation. A similar result was obtained by intramolecular disulfide
cross-linking between engineered pairs of cysteines in rhodopsin (40,
41).
Dissecting Specific Conformational Changes Involved in Receptor
Activation--
Rhodopsin activation has been analyzed by different
forms of spectroscopy including Fourier transform infrared resonance
spectroscopy (42, 43), surface plasmon resonance spectroscopy (44),
tryptophan UV absorbance spectroscopy (45), and EPR spectroscopy (41, 46, 47). All approaches have consistently provided evidence for
significant conformational rearrangements accompanying transition of
rhodopsin to metarhodopsin. Using tryptophan UV absorbance spectroscopy
Sakmar and co-workers (45) were able to obtain evidence that
photoactivation involves movement of TM3 relative to TM6. Further
insight into the character of conformational changes in rhodopsin has
been obtained by Khorana, Hubbell and co-workers (41, 46, 47) using EPR
spectroscopy in combination with multiple cysteine substitutions. By
site-selective incorporation of pairs of sulfhydryl-reactive spin
labels in a series of double cysteine mutants they were able to measure
changes in relative distance between TM3 and TM6 (41). The movement of
TM3 was interpreted as being relatively small whereas the data pointed
to a significant rigid-body movement of TM6 in a counterclockwise
direction (when the TM is viewed from the extracellular surface of the
receptor) and a movement of the cytoplasmic end of TM6 away from TM3
(41).
The use of fluorescence spectroscopic techniques in the
2 adrenergic receptor has allowed the first direct
structural analysis of conformational changes in a ligand-activated
GPCR (32, 48, 49). The spectroscopic technique used in these studies
takes advantage of the sensitivity of many fluorescent molecules to the
polarity of their local molecular environment (48). The sulfhydryl-reactive fluorophore
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ethylenediamine (NBD) was used to label free cysteine residues in purified,
detergent-solubilized
2 adrenergic receptor (48). Both
the quantum yield of the emission spectrum and the limited
accessibility to hydrophilic quenchers strongly suggested that one or
more of the naturally occurring transmembrane cysteines was labeled
with the fluorophore. Exposure of NBD-labeled receptor to agonist led
to a reversible and dose-dependent decrease in fluorescence
emission consistent with movements of the fluorophore to a more
hydrophilic environment following binding of the full agonist
isoproterenol (48). Interestingly, exposure of the NBD-labeled receptor
to inverse agonists led to an apparent increase in fluorescence,
suggesting that not only agonists but also inverse agonists can promote
structural changes in a GPCR (48).
Spectral analysis of a series of mutant
2 receptors
having only one, two, or three of the natural cysteines available for fluorophore labeling showed that NBD bound to Cys125 in TM3
and to Cys285 in TM6 was responsible for the observed
agonist-induced changes in fluorescence (49). This suggests that
movements of TM3 and TM6 occur during receptor activation (49). In a
molecular model of the
2 receptor based on the
projection map of rhodopsin, the NBD attached to Cys125 is
bounded by the lipid bilayer and the interface of TM3 and TM4, whereas
the NBD attached to Cys285 is bounded by TM6 and TM7 and
the lipid bilayer (49). In the framework of this model the change in
fluorescence of NBD-labeled
2 receptor can best be
explained by a counterclockwise rotation (when viewed from the
extracellular side of the membrane) of both TM3 and TM6, which would
move the NBD molecules from the nonpolar lipid environment to the more
polar interior of the protein (49).
In summary, both biophysical and mutagenesis studies of rhodopsin and a
number of ligand-activated GPCRs provide evidence that the formation of
the R* state involves movements of TM3 and TM6. Fig.
2B illustrates the type of movement that would be predicted
from the spectroscopic studies of rhodopsin and the
2
adrenergic receptor (41, 49). It should be noted that these studies do
not exclude the possibility that other TMs or cytoplasmic domains
undergo significant movement during activation. Further investigation
will be required to provide a more detailed map of changes in GPCR
structure during activation.
 |
Mechanism of Agonist Activation |
The Mode of Activation of Rhodopsin Is Unique among GPCRs--
As
discussed above, experimental evidence suggests that the conformational
changes associated with activation of rhodopsin are similar to changes
associated with activation of ligand-activated GPCRs such as the
2 adrenergic receptor. However, the remarkable structural diversity among GPCR agonists (small molecules, peptides, and proteins) and the apparent lack of a common agonist-binding site
suggest that the mechanism by which agonists induce the activating conformational changes in GPCRs may differ significantly for different receptors (5). Rhodopsin is unique among the GPCRs in that its ligand
is covalently bound to the receptor as an inverse agonist and upon
absorption of a photon isomerizes to an agonist within the binding
pocket (reviewed by Sakmar (50)). Thus, the process of ligand binding
is not part of the activation process. This specialized mechanism of
activation may be necessary to facilitate the very rapid response of
rhodopsin to light. Conversion from the inverse agonist
cis-retinal to the full agonist trans-retinal occurs within femtoseconds of photon activation. Rhodopsin then rapidly
undergoes a series of conformational changes that have been
characterized spectroscopically (rhodopsin > bathorhodopsin > lumirhodopsin > metarhodopsin I > metarhodopsin II). The
structural changes associated with the formation of metarhodopsin II,
the R* form of rhodopsin, are observed within microseconds of photoactivation even in detergent solution in the absence of transducin (46). Metarhodopsin II then undergoes a slow
(t1/2 ~6 min) transition to the inactive
metarhodopsin III (46). This inactivating transition is associated with
the hydrolysis and release of trans-retinal from the binding
pocket (51). It is interesting that free trans-retinal is
not a very effective agonist for opsin (52, 53), producing only ~14%
of the response observed for light-activated rhodopsin (52). Thus, the
efficient activation of rhodopsin by trans-retinal requires
that the agonist be rapidly generated by photoisomerization of the
prebound inverse agonist cis-retinal. The less efficient
activation of opsin by free trans-retinal may more closely
reflect the process of activation of other GPCRs.
In contrast to the extensive characterization of retinal
photoisomerization and the subsequent effects on the conformation of
rhodopsin, very little is known about the mechanism by which binding of
diffusible agonists such as catecholamines, peptides, and glycoprotein
hormones leads to the formation of the R* state. Recently it
has been possible to study agonist-induced conformational changes in
the human
2 receptor in real time by fluorescence
spectroscopy (32, 48, 49). These studies were done under conditions
that are similar to those used to study conformational changes in
rhodopsin (in the detergent
-dodecyl maltoside). In contrast to the
rapid activation and slow inactivation kinetics of rhodopsin, the
agonist-induced conformational changes observed in purified
2 adrenergic receptor are slow (t1/2 ~ 3 min) (48), even at agonist concentrations that would be predicted to ensure saturation of binding sites in less than 1 min (54). Reversal
of the agonist-induced conformation by antagonists is relatively rapid
(t1/2 ~30 s) (48). The differences in
activation kinetics between rhodopsin and the
2 receptor
may be influenced somewhat by the different methods used to monitor
conformational changes but most likely reflect fundamental differences
in the mechanism of activation by a covalently bound agonist
(trans-retinal) and a diffusible agonist
(isoproterenol).
Models of Activation by Diffusible Agonists--
The binding site
for catecholamines in the
2 adrenergic receptor is
remarkably similar to the binding site for retinal in rhodopsin (3,
50). The essential sites of interaction between catechol agonists and
the
2 adrenergic receptor are illustrated in Fig.
3A. Several models for the
interaction of ligands with their receptors have evolved from the study
of receptors, enzymes, and ligand-gated ion channels (55). The
2 adrenergic receptor is used to illustrate these models
in Fig. 3, B-D. "Ligand induction" (56), shown
mechanistically in Fig. 3B, predicts that transition from
the inactive to the active state is extremely rare in the absence of
agonist because of the energy barriers between R and
R*. The free energy of agonist binding to R is
used to overcome the energy barrier and facilitates (or induces) the
transition to R*. This model is consistent with the mechanism of activation of rhodopsin in that a photon rapidly converts
the inverse agonist cis-retinal to the agonist
trans-retinal, thereby inducing a rapid conformational
change in the protein. The model could also be used to explain the slow
agonist-induced conformational change observed for the
2
receptor by proposing a rapid association rate for agonist binding to
R and a slow rate for the transition from AR to
AR*. However, the model is inconsistent with the high basal
activity observed for many ligand-activated GPCRs, suggesting that the
energy barrier between R and R* is surmountable
in the absence of agonist.

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Fig. 3.
A, sites of
interaction between a catecholamine agonist and the
2 adrenergic receptor (reviewed in Ref. 3).
The amine (a) of agonists and antagonists interacts with an
aspartate in TM3 through a salt bridge. The aromatic catechol ring
(c) is thought to interact with a Phe290 in TM6.
There is also evidence for a functional interaction between the
-hydroxyl (b) and Tyr296 in TM6
of the 2 adrenergic receptor (60). Hydroxyl groups on
the catechol ring (d) have been shown to interact with
serines on TM5. B-D, models for the mechanism of agonist
activation of the 2 adrenergic receptor are discussed in
the text (B, ligand induction; C, conformational
selection; D, sequential binding and conformational
stabilization). Only TMs 3, 5, and 6 are shown to simplify the
illustration.
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The model shown in Fig. 3C will be referred to as
"conformational selection" and is based on the model proposed by
Koshland and Neet (57) and the extended ternary complex model for GPCRs (17). Transitions between R and R* can occur in the absence of agonists. Agonists bind preferentially to the
R* conformation and thereby shift the equilibrium and
increase the proportion of receptor in R*. Inverse agonists
bind preferentially to R and, therefore, reduce the
population of receptor in R*. This model can account for the
basal activity of GPCRs in the absence of agonist and explains the
action of inverse agonists. However, if the affinity of a full agonist
for R is assumed to be negligible, the model does not
readily accommodate the observation that the association rate for
agonist binding is very rapid whereas the kinetics of agonist-induced
conformational change in the absence of G protein is slow for the
2 adrenergic receptor. The model would predict that the
association rate for agonist binding is limited by the rate of
transition from R to R*, because agonist would
only bind to R*.
In limiting the number of conformational states to two (R
and R*), these models (Fig. 3, B and
C) fail to predict the observation that both agonists and
inverse agonists protect the
2 adrenergic receptor
against thermal denaturation (32) and proteolysis (58). These
experiments suggest that the unliganded receptor represents a distinct
state that is susceptible to denaturation and proteolysis, whereas the
conformations of the inverse agonist-bound state and the agonist-bound
state are both resistant.
Based on these observations a third model can be formulated (Fig.
3D). This model predicts that the unliganded receptor exists in a unique state R that can undergo transitions to at least
two other states, Ro and R*.
Ro is stabilized by inverse agonists, and
R* is stabilized by agonists. Mechanistically, this could be
explained by proposing that the unliganded receptor (R) is
not highly constrained by stabilizing intramolecular interactions and
is therefore more susceptible to thermal denaturation and proteolysis.
Moreover, R may undergo spontaneous transitions to the
R* state, explaining the high basal activity observed for
some GPCRs. As shown in Fig. 3D, binding of agonist domains
occurs sequentially, resulting in a series of conformational states
that are intermediates (R' and R") between
R and R*. As discussed above, results from
mutagenesis experiments indicate that
2 receptor agonists have several functionally important sites of interaction with
the receptor. Binding may involve an initial interaction between
receptor and one structural group of the agonist. Following the initial
binding of one structural group, binding of the remaining groups occurs
in a sequential manner as a result of random and spontaneous movements
of TM domains to positions that permit interaction with the functional
groups. Each interaction between the receptor and the agonist
stabilizes one or more TM domains until the receptor has been
stabilized in the active R* state. A similar mode of binding
can be envisioned for inverse agonists resulting in stabilization of
the Ro state. Partial agonists may stabilize one
of the intermediate states (R' or R"), thereby
increasing the chance of spontaneous isomerization to R*; or
they may stabilize unique conformational states having lower affinity
for the G protein. This model would be consistent both with a rapid
association rate for agonists (formation of AR') and the
relatively slow rate of conformational change observed
spectroscopically (formation of AR*). G proteins may
interact with the receptor to stabilize it in one of the intermediate
states (R' or R") and thereby influence both
agonist binding affinity and the kinetics of the conformational
change.
The model shown in Fig. 3 represents our best efforts to explain the
mechanism of GPCR activation with the limited experimental data
available. A more complete understanding of the molecular mechanism of
GPCR activation will require a high resolution structure, more detailed
information about the structural changes induced in the receptor by
different classes of ligands, and information from time-resolved
studies characterizing the sequence of conformational changes that
follow ligand binding.
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ACKNOWLEDGEMENTS |
We thank Pejman Ghanouni and Dr. Sansan Lin
for critical reading of the manuscript, and Jason Kobilka for
preparation of the figures.
 |
FOOTNOTES |
*
This minireview will be reprinted
in the 1998 Minireview Compendium, which
will be available in December, 1998. This is the second article of three in the "G
Protein-coupled Receptors Minireview Series." This work was supported
in part by National Institutes of Health Grant RO 1 NS28471 and by the
Harold G. and Leila Y. Mathers Charitable Foundation.
¶
To whom correspondence should be addressed: Howard Hughes
Medical Inst., B157 Beckman Center, Stanford University Medical School,
Stanford, CA 94305. Tel.: 650-723-7069; Fax: 650-498-5092; E-mail:
kobilka{at}cmgm.stanford.edu.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; TM, transmembrane; NBD,
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa1,3-diazol-4-yl)-ethylenediamine.
2
S. Rasmussen, P. Ghanouni, A. D. Jensen,
and U. Gether, manuscript in preparation.
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