Department of Pharmacology, University of Melbourne, Parkville,
Victoria, Australia (A.C.); and 7TM Pharmacology Systems Research,
Glaxo Smith-Kline Research and Development, Research Triangle Park,
North Carolina (T.K.)
G protein-coupled receptors (GPCRs) represent the
largest family of cell-surface receptors. These receptors are natural
allosteric proteins because agonist-mediated signaling by GPCRs
requires a conformational change in the receptor protein transmitted
between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary
complexes with other ligands or "accessory" proteins and display
altered binding and/or signaling properties in relation to the binary
agonist-receptor complex. Allosteric sites on GPCRs represent novel
drug targets because allosteric modulators possess a number of
theoretical advantages over classic orthosteric ligands, such as a
ceiling level to the allosteric effect and a potential for greater GPCR
subtype-selectivity. Because of the noncompetitive nature of allosteric
phenomena, the detection and quantification of such effects often
relies on a combination of equilibrium binding, nonequilibrium kinetic,
and functional signaling assays. This review discusses the development
and properties of allosteric receptor models for GPCRs and the
detection and quantification of allosteric effects. Moreover, we
provide an overview of the current knowledge regarding the location of
possible allosteric sites on GPCRs and candidate endogenous allosteric
modulators. Finally, we discuss the potential for allosteric effects
arising from the formation of GPCR oligomers or GPCRs complexed with
accessory cellular proteins. It is proposed that the study of
allosteric phenomena will become of progressively greater import to the
drug discovery process due to the advent of newer and more sensitive GPCR screening technologies.
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I. Introduction |
A general property of all receptors is the ability to interact
with their endogenous ligands (hormones and neurotransmitters) to alter
cellular responsiveness without changing the chemical nature of the
ligand. This is in contrast to enzymes, where oftentimes a substrate is
made to bind in an energetically unfavorable mode that leads to its
eventual modification. G protein-coupled receptors (GPCRs) constitute the largest
superfamily of receptors and, not surprisingly, mediate the majority of
transmembrane signal transduction in living cells. These receptors
respond to a wide range of relatively small and structurally diverse
chemicals such as biogenic amines, peptides, hormones, and even light
with global changes in receptor conformation that then lead to larger
scale protein-protein interactions.
Traditionally, the unifying feature of GPCRs has been their interaction
with G protein(s) to transduce stimuli imparted to the receptor from
the extracellular environment to the intracellular response machinery
of the cell. Implicit in this mechanism, therefore, is the fact that
the intracellular contact points on the GPCR recognized by the G
protein are necessarily distinct from the extracellular domains used by
endogenous ligands. The lateral translocation of GPCRs in the cell
membrane to interact with their cognate G protein(s) is the best known
example of GPCR-protein interaction, but it is by no means the only
such example, because additional protein coupling partners are now
being rapidly identified for the GPCR superfamily (vide infra). The
entire surface of a GPCR can be considered a potential binding site for
biologically active molecules, both proteins and small molecules such
as drugs. It is a major premise of this review that a tripartite system composed of a ligand, a GPRC, and an additional GPCR coupling partner
represents a general motif for ligand action at GPCRs extending beyond
the G protein example. In other words, the requisite interaction
between topographically distinct binding sites on a GPCR to effect
change in cellular function identifies these receptors as natural
allosteric proteins.
Drugs have traditionally been discovered through the screening of
numerous chemical structures on a biological system. The greater the
number of structures tested, the greater is the probability of
detecting a biologically active ligand. Throughout this process, it is
clear that the type of receptor screen employed to detect biologically
active molecules will greatly define the types of molecules detected.
Thus, if the tracer molecule in the screen is a radioligand, then the
ligands most readly detected by that screen will be those that obstruct
the access of the radioligand to its specific binding site. Notably,
the current emphasis away from radioligand binding and toward high
throughput functional screening is beginning to reveal ligands that can
change biological function without exerting apparent effects on
radioligand binding. It is possible that such ligands are not
interacting with the classic, agonist-binding domain of the receptor
but rather with other topographically distinct domains.
This raises an interesting philosophical point in drug discovery,
namely the current paucity of allosteric ligands in the known
population of biologically active molecules. On one hand it could be
assumed that this paucity reflects their relative unimportance and
rarity in chemical space. However, another point of view would suggest
that this paucity reflects the bias imposed on the drug screening
process through the use of radioligand binding. As outlined above, the
need for high throughput screening has, in the past, required
radioligand binding assays to achieve the required volume of sampling
of chemical space for drug discovery. However, the improved technology
of functional screening in the new millennium will certainly test the
potential effects of this bias because the throughput available for
functional testing in reporter, yeast, and melanophore systems now
equals and in many cases surpasses that of radioligand binding. In
turn, this increased screening capability should cause an increase in
the texture of biologically active molecules detected. Whereas, before
1995, the primary chemical targets were agonists, partial agonists, and
antagonists, the availability of functional screens should allow the
detection of new classes of drugs. For example, allosteric enhancers
potentiate the effects of agonists either through enhancement of
agonist affinity, stabilization of agonist/receptor and G protein interaction or other unspecified enhancement of efficacy (vide infra).
Similarly, allosteric modulators could block agonist stimulation of the
receptor without necessarily interfering with agonist binding to the
receptor. Allosteric agonists could activate receptors without being
subject to appreciable blockade by classic antagonists. This review
will discuss examples of these types of ligands and the different
manifestations of allosterism at GPCRs.
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II. Allosteric Receptor Models of G Protein-Coupled Receptors |
A. Historical Perspective
Most of the theoretical framework associated with the study of
ligand-receptor interactions was developed in the first half of the
twentieth century, when very little was known about the actual identity
of receptors themselves. By borrowing from studies in the field of
enzyme kinetics, pharmacologists and physiologists adopted the law of
mass action as a minimal mechanistic descriptor of the interaction
between a ligand and its receptor. Often, the simplest form of the mass
action model
a reversible, saturable, one-to-one interaction between
ligand and receptorwas deemed compatible with experimental
observations. Even today, where much has been accomplished in terms of
identifying the proteinaceous nature and molecular properties of the
major receptor families, the starting point for the qualitative or
quantitative analysis of drug-receptor data remains the concept of the
drug interacting at a "primary" binding site recognized by agonists
and competitive antagonists.
The classical view of ligand-receptor interactions mentioned above has
served pharmacologists faithfully in studies of receptor mechanisms,
classification, and drug discovery, yet as early as the 1930s one of
the pioneers of analytical pharmacology, A. J. Clark (1937)
,
postulated the existence of a "complex receptor with which one drug
can unite without displacing the other drug". In an extensive
treatise on drug-receptor theory, Ariëns et al. (1956) formalized
and extended Clark's speculation by developing a mathematical model
for a noncompetitive interaction between "a substance A and a
receptor system R, the latter being partly inactivated or sensitized as
a result of the interaction of a substance B with another receptor
system". In Ariëns' model, both "receptor systems" were
considered to be interdependent, "possibly representing two distinct
active loci on the one protein molecule". In a similar vein, Van den
Brink (1969) coined the term "metaffinoid antagonism" to define
potential drug-receptor interactions where a change in the binding site
of the antagonist led to a change in the binding site of the agonist,
resulting in a subsequent reduction in agonist affinity for its
receptor. Hence, the concept of cross-interactions between the agonist
binding site and other potentially distinct binding domains on
receptors was a relatively early, albeit mainly theoretical, component
of classic receptor theory, alongside the better-known and by far better-studied concept of competitive drug-receptor interactions (Gaddum, 1936; Arunlakshana and Schild, 1959; Kenakin, 1997c).
Much of the early drug-receptor theory was developed to describe the
behavior of receptors that would later be identified as GPCRs.
Unfortunately, detailed mechanistic studies on these receptors were
initially hampered by the fact that the requisite dissociation of the
ligand-receptor binding process from the subsequent signal transduction
events that characterize GPCR activity meant that there were no
sufficiently detailed tools with which to dissect drug actions at these
receptors at the molecular level. This meant that for some time,
drug-GPCR theory remained largely operational. In contrast, early
studies of enzymes and voltage- and ligand-gated ion channels did not
suffer from the same drawbacks as their GPCR counterparts and, thus,
the two most important mechanistic insights that led directly to the
current models of allosterism at GPCRs were derived from the enzyme and
ion channel arena.
1. Cooperativity in Binding.
The first important development
in allosteric theory came from experimental evidence indicating that
more than one molecule of ligand was able to bind to certain enzymes or
ion channels to effect a change in the properties of the protein, a
phenomenon termed "cooperativity". In fact, the well known Hill
equation commonly used nowadays to empirically fit
concentration-response data was originally derived to describe
cooperative binding (Hill, 1910). Figure
1 illustrates two classic examples of
cooperative binding proteins, the enzyme hemoglobin and the
GABAA ion channel-receptor complex. Simple
mass-action kinetics predict that the binding of a single molecule of
ligand to a single binding site on a protein would yield a hyperbolic
isotherm (when plotted on a linear scale) with a slope coefficient
equal to unity. However, the binding of oxygen to hemoglobin (Fig. 1A)
or GABA to the GABAA receptor (Fig. 1B) are
characterized by distinctly sigmoid curves when plotted on a linear
scale, reflecting the multiple equivalents of ligand binding to the
same protein complex. Studies such as these conducted on a variety of
ion channel-linked receptors, thus, led to the conclusion that certain
receptors can possess more than one binding site for ligands. This
concept invoked another phenomenon that was also originally described
in the field of enzymology, that is, the idea of allosteric (or
allotopic) binding sites.
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Fig. 1.
Cooperative binding in enzymes and ion
channel-linked receptors. A, the binding of oxygen to hemoglobin dimers
(curve D, Hill slope = 1) and tetramers (curve T, Hill slope = 3.3). Concentrations of hemoglobin range from 40 nM (D) to 100 µM
(T). Data taken from Ackers et al. (1992). B, conductance change at the
crustacean neuromuscular junction produced by  -aminobutyric acid
(GABA). Redrawn from Colquhoun (1973) based on data of Takeuchi and
Takeuchi (1969).
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The term "allosteric" (from the Greek meaning "other site") was
first used by Monod and Jacob (1961) and subsequently defined by Monod
et al. (1963) in a paper describing the ability of enzymes to have
their biological activity modified, in either a positive or negative
fashion, by the binding of ligands to sites that were topographically
distinct from the substrate-binding site. Monod et al. (1963) defined
these accessory binding sites as allosteric sites, in contrast to the
substrate-binding (active) site, which was defined as the isosteric
site. In their original paper, Monod et al. (1963) outlined three
general classes of interactions between two ligands on the one enzyme
molecule. Class I interactions represented classic competition, where
the substrate and inhibitor competed for overlapping regions on the
receptor. Class II interactions were deemed to encompass situations
where an inhibitor could form an attachment with a region of the enzyme
not recognized by the substrate while some of the inhibitor molecule
could interact with the substrate-binding site in a competitive manner.
An example of this type of "direct interaction" nowadays is the
effect of the "captive agonist" salmeterol at the
2-adrenoceptor, where the long alkyl side
chain of the molecule forms a persistent attachment with the receptor
that allows its salbutamol-like active moiety to interact with the
classic agonist binding domain to yield a persistent response (see
Coleman et al., 1996). The final type of interaction (class III) was
termed "indirect" or "allosteric". These interactions arise
when the binding of a ligand to the allosteric site induces a
conformational change in the protein and modulates the binding of the
substrate to the isosteric site, and vice versa. The biological
activity of the enzyme was subsequently assumed to arise from the
modified properties of the substrate-binding site, and not through a
direct effect of the allosteric modulator itself. Monod et al. (1963)
referred to this conformational change in the enzyme as an
allosteric transition, although that term has since come to
encompass a slightly different concept (see below).
With regards to receptor proteins, the primary binding site recognized
by the endogenous agonist or hormone is conceptually equivalent to an
enzyme's isosteric site, and has been referred to as the orthosteric
site (Proska and Tucek, 1994; Christopoulos, 2002). Any binding site on
a receptor protein that is able to modulate the binding properties of
the orthosteric site by mediating a conformational change in the
receptor may be classed as an allosteric site. Hence, many of the
cooperative interactions that had been reported for ion channel-linked
receptors in the literature in the past, such as the binding of two
acetylcholine molecules to a single nicotinic acetylcholine receptor
(Galzi et al., 1991) or the binding of two GABA molecules to a
GABAA receptor (Sigel and Buhr, 1997), are also
allosteric interactions because the binding of one equivalent of ligand
actually alters the affinity of the subsequent binding of the next
equivalent(s) of ligand.
2. Allosteric Transitions: Multistate Models of Receptor
Action.
Before discussing allosteric mechanisms in greater
detail, it is necessary to address some of the issues that have arisen in the past regarding the terminology applied to allosteric proteins (Table 1). The term "allosteric" has
been used by a number of authors in different ways, and this has led to
some confusion in the literature as to what it actually means (e.g.,
see Colquhoun, 1998). Nowadays, it seems that a distinction is
necessary between the terms "allosteric interaction" and
"allosteric transition". For the purposes of this review, an
allosteric interaction is defined as an interaction that occurs between
two (or more) topographically distinct binding sites on the same
receptor complex. The essential features of a simple allosteric
interaction are as follows: (a) The binding sites are not overlapping,
that is, there is no mutual exclusivity in binding. (b) The binding of
one ligand to its site affects the binding of the second ligand at the
other site and vice versa. Allosteric interactions are, thus,
reciprocal in nature. (c) The effect of an allosteric modulator can be
either negative or positive with respect to the binding and/or function
of an orthosteric ligand.
Although Monod et al. (1963) initially defined the conformational
change in protein structure associated with an allosteric interaction
as an allosteric transition, they subsequently presented a more
formalized model of allosteric proteins that gave rise to the second
major development in allosteric theory, namely, an emphasis away from
interactions occurring between sites to interactions occurring between
conformational states (Monod et al., 1965). Allosteric proteins were
then described by these authors as follows: (a) They are oligomeric in
nature (i.e., composed of more than one subunit). (b) Each subunit
possesses one (equivalent) binding site for ligand, thus, giving rise
to cooperative interactions. (c) They can exist as an equilibrium
mixture of two or more states in the absence of ligand, with the
transition between states now being defined as the allosteric
transition. (d) The transition between conformational states involves a
conservation of molecular symmetry such that all subunits "flip"
from one state to another in a concerted fashion. (e) Ligands that
prefer binding to one state over another will "select" the
preferred state and, thus, increase the proportion of proteins in that
state. As a consequence, observed (macroscopic) ligand affinity will
alter depending on the type and amount of conformational state that predominates.
It can be seen that this last definition of allosteric proteins is
quite explicit. Its description of interactions between multiple
subunits makes it immediately applicable to oligomeric proteins that
display cooperative binding, e.g., ion channel-linked receptors. It
should be noted that models dealing with receptor isomerization between
different conformational states were published as early as the 1950s to
describe the postulated mechanism of action of the nicotinic
acetylcholine receptor (del Castillo and Katz, 1957; Katz and Thesleff,
1957), although the actual term allosteric was not coined until the
subsequent work of Monod and colleagues (1963). An important property
of receptor models that incorporate allosteric transitions between
conformational states is the prediction of receptor activity in the
absence of ligand as a consequence of the isomerization process, i.e.,
constitutive receptor activity (Karlin, 1967; Colquhoun, 1973; Thron,
1973; Leff, 1995). These models are now more commonly referred to as "two-state" or "multi-state" models and represent the simplest mechanism approximating certain known aspects of protein behavior. In
essence, the two-state model of receptor action is a mechanism of
conformational selection, whereby a ligand selectively binds to a
pre-existing receptor conformation, thereby creating a bias toward that
conformation. In terms of free energy, this mechanism is generally
preferable to one of conformational induction, where the ligand
actually creates the conformation through the binding process (Burgen,
1981; Kenakin, 1995a). It should be noted, however, that conformational
selection and conformational induction most likely represent two
extremes of a common mechanism used by proteins in changing the type
and abundance of conformational state in the presence of ligand.
On the surface, the concept of receptor allosterism within the context
of multiple conformational equilibria may seem somewhat removed from
the concept of an interaction occurring between distinct binding sites
on the one protein. For instance, multistate models allow allosterism
to arise simply as a consequence of the transition between one
orthosteric conformation to another, without necessarily postulating
the existence of a second binding site in each conformational state. In
contrast, the simple model of allosteric interaction between two sites
does not explicitly consider the existence of multiple conformations of
the protein on which the sites are situated. As will be discussed
below, these two ideas are not mutually exclusive; rather they address
different aspects of a protein's ability to undergo conformational
changes. To avoid engendering further confusion, the remainder of this
review will use the term "receptor isomerization" when describing
the transition of receptors between multiple conformational states and
"allosteric interaction" when describing a reciprocal interaction
between multiple binding sites on the same protein.
3. Allosteric Interactions: Ternary Complex Models.
Ion
channels and ion channel-linked receptors are known to exist as
oligomers; that is, they are composed of multiple protein subunits, and
with an increased complexity in macromolecular structure comes an
increased probability of multiple ligand binding sites. Allosteric
interactions at ion channel-linked receptors, therefore, have been well
documented and studied for almost half a century now. In contrast,
GPCRs have, until recently, been considered traditionally to exist as
monomers, and relatively fewer allosteric interactions occurring at
GPCRs have been identified relative to ion channel-linked receptors.
Nevertheless, it is now apparent that orthosteric ligand binding at
GPCRs can be subject to allosteric modulation by other ligands or other proteins.
The best known example of an allosteric modulator of ligand binding to
GPCRs is the G protein itself, and, as with the original formulation of
allosteric theory in relation to enzymes and ion channels, the
development of the current allosteric models for GPCRs was also based
on two major ideas. The first idea was the development of two-state
theory for ion channels and ion channel-linked receptors, as described
above (del Castillo and Katz, 1957; Katz and Thesleff, 1957; Karlin,
1967; Colquhoun, 1973; Thron, 1973; Leff, 1995). These models described
how selective affinity of ligands for specific receptor states (in the
case of either open or shut ion channels) could bias the system toward
the favored state. The second major idea in the GPCR field was that
receptors could translocate within membranes and associate with other
membrane-bound proteins (Cuatrecasas, 1974). Thus, any mechanism
ascribed to a GPCR would need to explicitly invoke the presence of at
least two binding sites on the same receptor protein, one for the
orthosteric ligand and one for the G protein. This tripartite coupling
mechanism represents the simplest scheme for an allosteric interaction
occurring between distinct sites (as opposed to states) on a single
receptor protein.
In general, the interaction between agonist binding and G protein
coupling is positively cooperative in nature (Ehlert, 1985). This is
logical, given the mechanisms that are thought to underlie signaling
via GPCRs (Gilman, 1987; Bourne, 1997; Hamm, 1998). Agonist binding to
the orthosteric site results in an alteration of receptor conformation
that displays a higher affinity toward the G protein, thus favoring
coupling. However, the binding of GTP to its site on the G protein
results in a change of G protein structure that is transmitted to the
receptor's conformation as a negatively cooperative effect on agonist
binding, thus promoting the uncoupling of the activated G protein from
the receptor and allowing signaling to proceed. These negatively
cooperative effects of GTP on agonist binding underlie the so-called
"GTP shift" that has often been used as a biochemical measure of
agonist efficacy (Kenakin, 1997c; Christopoulos and El-Fakahany, 1999).
Figure 2 summarizes the evolution of GPCR
models from simple operational schemes to the contemporary ternary
complex mechanisms. The original TCM, as described by De Lean et al.
(1980) allowed a ligand-bound activated receptor to form a G protein
complex resulting in activation. This is a simple example of a receptor isomerization mechanism, where the binding of ligand A promotes a
conformation of receptor that either signals in its own right (e.g.,
ion-channels; Fig. 2A, left) or couples to and activates a G protein
(Fig. 2A, right). The next level of progression toward present GPCR
models also involved the incorporation of different receptor
conformations into the GPCR scheme. This latter development owed much
to the introduction of recombinant receptor systems into receptor
pharmacology, because it allowed for the ability to control the
stoichiometry between receptors and G proteins. With this capability
came the discovery of constitutive GPCR activity due to the spontaneous
coupling of receptors in active conformations to G proteins in the
absence of ligands. For this to occur, the minimal receptor model for
such a system is shown in Fig. 2B (left). In the figure, L is the
isomerization constant defining the equilibrium between active (R*) and
inactive (R) receptor states, Ka is
the equilibrium association constant of the ligand-receptor complex and
is referred to as a "cooperativity factor", i.e., it is a ratio
of the affinity of the ligand for the active versus the inactive state
of the receptor. Alternatively, it may be viewed as a measure of the
ability of ligand-bound receptor to enrich the R* state. The use of
cooperativity factors in closed equilibrium reaction schemes such as
those shown in Fig. 2 serves to reduce the number of parameters
required to describe a model while satisfying the principle of
microscopic reversibility (Wyman and Allen, 1951; Weber, 1975; Wyman,
1975; Ehlert, 1985; Weiss et al., 1996a). This idea, also referred to
as the concept of "free energy coupling" (Weber, 1972, 1975),
states that the energy required to reach one species from another must
be the same at equilibrium, irrespective of what path is chosen, hence,
the use of the cooperativity factor .
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Fig. 2.
The evolution of allosteric receptor models for
GPCRs. The earliest models were based on the assumption that the law of
mass action dictates the binding of ligand A to the receptor, R,
according to the equilibrium association constant,
Ka, and then subsequently resulted in a
response. This operational approach was then impacted upon by a
progression of mechanistic insights. A, the agonist bound receptor can
isomerize to produce a different state that can signal on its own
(left) or translocate within the membrane to interact with a G protein
(right). B, the receptor, R, can spontaneously isomerize to an active
state, R*, (left) or couple to a G protein, G, or allosteric ligand, B,
(right) in the absence or presence of orthosteric ligand. Thermodynamic
considerations dictate that the isomerization constant, L, and the
equilibrium association constants, Ka,
Kb, and Kg, are
modified to an extent governed by the cooperativity factors, , ,
or  , when the same interactions take place on an occupied receptor.
C, the ETC model of Samama et al. (1993) combines the two-state model
with the ternary complex model but only allows for the active receptor
state to interact with G protein. D, the CTC model (left) of Weiss et
al. (1996a, 1996b, 1996c) allows the inactive R state to interact with
G protein and the active state. This model is formally identical with
the allosteric two-state model (right) of Hall (2000), which describes
the interaction of an allosteric modulator and orthosteric ligand on a
receptor that can adopt active and inactive conformations.
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When developing the original TCM, De Lean et al. (1980) also considered
the possibility of a closed (cyclic) system operating in equilibrium,
that is, they speculated about the existence of precoupled RG complexes
in the absence of bound ligand (Fig. 2B, right). However, direct
evidence for this phenomenon was lacking at the time and had to be
inferred from the analysis of complex radioligand binding isotherms.
Nevertheless, the proposal of a requisite ternary complex mechanism to
account for the known behavior of GPCRs paved the way for further
explorations into the properties of such a model (Wregget and De Lean,
1984; Ehlert, 1985). Importantly, the symmetry of the model allowed it
to be equally applicable to situations where more than one type of drug
molecule could occupy the receptor at the same time (Stockton et al.,
1983; Ehlert, 1988). Observations made initially on studies of the
actions of a series of hexamethonium derivatives and the neuromuscular
blocking agent gallamine on muscarinic acetylcholine receptors had
already suggested that such a mechanism may be operative (Lüllman
et al., 1969; Clark and Mitchelson, 1976; Stockton et al., 1983). Thus,
the simultaneous binding of an orthosteric ligand, A, and an allosteric
ligand, B, to the receptor would be governed by the respective
equilibrium association constants, Ka
and Kb, just like the binding of an
orthosteric ligand and G protein would be governed by the constants
Ka and
Kg (Fig. 2B, right). As before with
the closed two-state model, the thermodynamic requirement of
reversibility also adds cooperativity factors to the affinities between
receptor, orthosteric ligand, and allosteric ligand () or G protein
() in the full ternary complex model. Interestingly, this principle
is common in most applications of allosteric theory and stems from the
idea that, as described by Sir Francis Bacon in 1620 "it is certain
that all bodies whatsoever have perception"; in terms of the ternary
complex model for receptors, if a receptor species is bound to some
other species in the system, then it cannot be considered identical
with its unbound counterpart. For example, if the receptor is bound to
ligand, its affinity for G protein is
Kg not
Kg. If it is bound to another ligand,
B, then its affinity for agonist is
Ka and not
Ka. This form of the TCM was the first
explicit model of allosteric interactions occurring between
topographically distinct binding sites applied to a GPCR, and it is
still a useful, minimal model with which to assess and quantify
experimental data (vide infra). It should be noted, however, that the
TCM as an allosteric model of receptor-G protein interactions,
on one hand, and receptor-modulator interactions, on the other, can
lead to different predictions with respect to the binding curve of the
orthosteric ligand. This is because G protein accessibility to
receptors within the plane of the membrane can often be limiting,
leading to shallow and/or biphasic orthosteric ligand binding curves
due to G protein depletion (see Ehlert, 1985). In contrast, allosteric
modulator drugs, like orthosteric ligands, are invariably present in
vast excess relative to the concentration of receptor, and ligand
depletion is, thus, much less likely to occur; the simple TCM
does not predict biphasic or shallow binding curves in the absence of
ligand depletion (vide infra).
The subsequent conclusive demonstration of constitutive GPCR activity
by Costa and Herz (1989) indicated that receptors could couple to and
activate G proteins in the absence of ligand. This necessitated the
modification of the original TCM described by De Lean et al. (1980),
which did not have the capability of spontaneous formation of the R*G
species, into the extended ternary complex model (ETC model; Samama et
al., 1993), as is shown in Fig. 2C. From this scheme, it can be seen
that the amount of active-state receptor available for subsequent
coupling to G protein is given by the isomerization constant L. Therefore, increasing the relative stoichiometry of receptors versus G
protein leads to an elevated abundance of R*G, the species responsible
for agonist independent response (constitutive receptor activity). For
example, for a system containing 1000 receptors and a value for L of
0.001, there will be one single R* species. However, if the receptor
number were to be increased by a factor of 1000, then the number of
receptors in the signaling R*G form would be 1000. By increasing the
number of receptors present in the system, the number of spontaneously active receptors can be increased until a threshold is attained where
the resulting response from the spontaneously formed R*G species can be
observed. The ETC model was, thus, the first GPCR model to explicitly
incorporate allosteric transitions between receptor states (e.g.,
governed by L and ) and allosteric interactions between multiple
binding sites (e.g., governed by and ).
Although the ETC model went beyond the original ternary complex model
to accommodate experimental findings, it is thermodynamically incomplete. Again, this is directly related to the principle of free
energy coupling described above, and has culminated in the development
of the more thermodynamically complete, albeit more complex, cubic
ternary complex (CTC) model by Weiss et al. (1996a-c; Fig. 2D, left).
Although the CTC model is formally more correct than the ETC model,
this correctness comes at a price of carrying too many parameters to
allow for useful estimation based on experimental observations. In
turn, this can make the model less predictive. Therefore, in practical
terms, it is worth considering whether the more complex CTC model is
worth applying to experimental data instead of the ETC model. The
critical issue is the need for the ARG complex, the nonsignaling
ternary complex between ligand, receptor, and G protein.
There are two approaches that can be used to try to determine which
model, ETC or CTC, has greater utility in the receptor pharmacology of
GPCR systems. One is the biochemical evaluation of the evidence for the
existence of the inactive ARG complex. To date, there is a paucity of
such evidence but it is not clear whether this is because of the
apparent rarity of this species in biological systems or because of the
lack of tools for detecting this species. There are isolated cases
where experimental data are consistent with the existence of a
nonsignaling ternary complex species. One example involves the inverse
agonist ICI-174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH)
acting at the Gi/o-coupled 
-opioid receptor
expressed in HEK 293 cells (Chiu et al., 1996). Whereas the opioid
agonist DPDPE mediated an inhibition of forskolin-stimulated cAMP
accumulation, ICI-174,864 caused a further stimulation of the cAMP
response above the basal forskolin response, consistent with the
inverse agonist properties previously ascribed to ICI-174,864 (Costa
and Herz, 1989). However, pretreatment of the cells with pertussis
toxin, which uncouples Gi/o-proteins from their
receptors, resulted in an abolition of both the agonistic effects of
DPDPE and the inverse agonist effects of ICI-174,864. Although the
former finding is consistent with the expectation that agonists require active receptor-G protein complexes, the latter finding with
ICI-174,864 is inconsistent with the notion that inverse agonists
prefer uncoupled receptor-G protein complexes to promote a reduction in
constitutive receptor activity. One explanation for the pertussis toxin
sensitivity of the ICI-174,864 effect is the possibility that this
particular inverse agonist attenuates constitutive receptor activity
not by uncoupling receptor-G protein complexes, but rather by promoting a stable ARG complex that is unable to signal.
Another example of a possible nonsignaling ARG ternary
complex involves the cannabinoid CB1 receptor,
where the inverse agonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide decreased constitutive receptor activity (as measured by activation of
mitogen-activated protein kinase) according to standard inverse agonist
kinetics for the receptor but also, unexpectedly, blocked the pertussis
toxin-sensitive activation of the same kinase by insulin (Fig.
3A) and insulin-like growth factor 1 receptors (Bouaboula et al., 1997). The crossover inhibition was
dependent on the presence of the CB1 receptor and
did not occur with the non-GPCR, fibroblast growth-factor receptor.
Crossover inhibition was also observed when Mas-7 (a mastoparan analog)
was used to directly activate Gi/o proteins and
suggests that G protein "trapping" was operative through the
interaction between SR141716A and CB1 receptors
to make Gi/o protein inaccessible to other
receptor pathways. This suggests the existence of the nonsignaling ARG
species in this receptor system.
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Fig. 3.
Biochemical evidence for a nonsignaling [ARG]
ternary complex. A, interaction of the inverse agonist, SR141716A, with
the cannabinoid CB1 receptor abolishes
Gi/o-dependent mitogen-activated protein kinase signaling
mediated by the insulin receptor tyrosine kinase, possibly by
sequestering G protein in an inactive ternary complex of
inverse-agonist, CB1 receptor, and G protein. Data taken
from Bouaboula et al. (1997). B, dissociation kinetics of opioids in
CHO cell membranes expressing the human µ-opioid receptor. Unlike the
antagonist [3H]diprenorphine, the antagonist
[3H]NalBzOH and the agonist [3H]DAMGO each
displayed biphasic dissociation kinetics, indicative of two affinity
states of the receptor. The biphasic binding was sensitive to guanine
nucleotides, suggesting that both [3H]DAMGO and
[3H]NalBzOH were coupling to G proteins, but only the
former agent was able to initiate a response. Data taken from Brown and
Pasternak (1998).
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Similarly, in CHO cells stably transfected with µ-opioid receptors,
there is biochemical evidence of a nonsignaling ligand/receptor/G protein complex. In this system the potent µ-opioid receptor
antagonist naloxone benzoylhydrazone (NalBzOH) blocks agonist-mediated
cyclic AMP responses. However, a 3-fold enhancement of affinity was
observed for NalBzOH in equilibrium binding studies in the presence of the stable GTP analog Gpp(NH)p. This indicated a low level of negative
efficacy for this ligand at this receptor and also that NalBzOH has a
preferential affinity for the inactive state of the receptor. In
apparent contrast to this, [3H]NalBzOH
demonstrated biphasic kinetics indicative of two affinity states (Fig.
3B), consistent with an association of at least one state with G
protein (Brown and Pasternak, 1998). An association with G protein
(with no concomitant signaling) was indicated by the elimination of the
high affinity state by Gpp(NH)p. The lack of a similar effect by the
µ-opioid antagonist diprenorphine and the production of this same
effect with pertussis toxin treatment indicated that the high-affinity
component was a ligand-specific receptor complex associated with
Gi/o protein.
Most recently, a study by Chen et al. (2000a) provided strong evidence
for the potential of a mammalian GPCR to inhibit signaling in a
dominant-negative manner by sequestering G protein -subunits in a
nonsignaling ternary complex. Specifically, a point mutation in Phe303
in the sixth transmembrane domain of the
1b-adrenoceptor resulted in a receptor that
displayed enhanced agonist binding affinity relative to the wild type,
but a loss in agonist-mediated signaling through the phosphoinositide
(PI) pathway. Furthermore, the mutant receptor, but not the wild type,
could be coimmunoprecipitated with Gq in the
absence of agonist, indicating a tight coupling of mutant receptor to G
protein, and overexpression of Gq-subunits resulted in a rescue of the dominant negative activity of the mutant
with respect to PI signaling. Taken together, these findings are
compatible with the ability of the mutant
1b-receptor to selectively sequester
Gq-subunits in a conformation that promotes high agonist binding affinity but not signaling.
A second potential method of determining which model best fits a given
experimental system is to examine the predictions of the models and
compare those with experimental findings. For example, both the ETC and
CTC models predict that increasing the amount of G protein available to
the receptor will increase the amount of R*G species and, subsequently,
the amount of constitutive activity. The relationship between G protein
and constitutive activity predicted by the ETC model is given by (Chen
et al., 2000b)
![<FR><NU><UP>basal</UP></NU><DE><UP>max</UP></DE></FR>=<FR><NU><FR><NU>&bgr;<UP>L</UP>[<UP>G</UP>]</NU><DE>K<SUB><UP>G</UP></SUB></DE></FR></NU><DE>1+<FR><NU>&bgr;<UP>L</UP>[<UP>G</UP>]</NU><DE>K<SUB><UP>G</UP></SUB></DE></FR></DE></FR>](/content/vol54/issue2/fulltext/323/img001.gif)
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(1)
|
As can be seen from the above equation, at theoretically infinite
concentrations of G protein, the constitutive activity will reach the
system maximal response. A different relationship is predicted by the
CTC model. As described by Weiss et al. (1996a,b,c), the relationship
between constitutive activity and receptor number, expressed as a
fraction of the maximal system response, is given by
![<FR><NU><UP>basal</UP></NU><DE><UP>max</UP></DE></FR>=<FR><NU><FR><NU>[<UP>R</UP>]</NU><DE>K<SUB><UP>G</UP></SUB></DE></FR>(1+&dgr;&agr;&bgr;<UP>L</UP>)</NU><DE><AR><R><C> </C></R><R><C>&dgr;&agr;<FENCE>1+<FR><NU>[<UP>R</UP>]</NU><DE>K<SUB><UP>G</UP></SUB></DE></FR>(1+&bgr;<UP>L</UP>)</FENCE></C></R></AR></DE></FR>](/content/vol54/issue2/fulltext/323/img002.gif)
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(2)
|
If receptor concentration is not limiting (i.e., as [R] 
), then the constitutive activity will reach an asymptotic value of
|
(3)
|
For a high-efficacy agonist, 
1 and the expression
reduces to
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(4)
|
Due to the possibility of producing a nonsignaling RG species, the
CTC model predicts that the constitutive activity produced by addition
of G protein need not reach the system maximum.
It can be seen that the two models predict the same qualitative but
differing quantitative responses. Unfortunately, although submaximal
levels of constitutive activity have been observed with receptor
transfection experiments in Xenopus laevis melanophores (Chen et al., 2000b), it is not possible to determine whether the G
protein levels in these cells were limiting and, thus, prevented the
production of system maximal response. Also, because cellular responses
are amplified functions of [R*G], it is not possible to determine
whether a full constitutive maximal response relates to a submaximal or
fully maximal conversion of receptor species to R*G.
It is presently unclear which of these models better predicts and
describes experimental findings with GPCRs. On the practical side, the
ETC model has fewer parameters, is simpler to use, and is, therefore,
parsimonious. The CTC model is heuristic and more encompassing but has
a greater number of nonestimatable parameters. It could be that
different systems are better suited for different models, i.e., there
may be GPCR systems where the ARG is so unimportant as to be
negligible, thereby, making the ETC model preferable, and other systems
where the ARG species plays a role, thus, necessitating use of the CTC model.
Another application of the CTC model exploits the symmetry in the model
with respect to the reciprocity of interaction between orthosteric and
allosteric sites. If it is assumed that the ligand occupying the
secondary site on the receptor is not a G protein, but rather an
allosteric modulator drug, then the model can be recast to yield a
mathematical description of drug-drug allosteric modulation between two
binding sites on a receptor that exists in both active and inactive
states (see Fig. 2D, right). The properties of this "allosteric
two-state model" were recently explored by Hall (2000), who compared
it to the CTC model for agonist-G protein interaction. Although the
equations derived from the model are formally identical with those of
the G protein-based CTC model, there are important differences between
the two models with respect to the effects of the cooperativity factors
on receptor activation (Hall, 2000). This is because the allosteric
two-state model (Fig. 2D, right) quantifies response as the production
of activated receptor species (R*, AR*, BR*, and AR*B), as would be the
case for ion channel-linked receptors. In contrast, the CTC model
quantifies response as the production of activated receptor-G protein
species (i.e., R*G, AR*G). Thus, the parameter in the allosteric
two-state model only modifies orthosteric ligand affinity; the
equivalent parameter in the CTC model, , modifies the ability of
agonist to interact with G and, thus, affects response production and efficacy. As with the CTC model versus the ETC model, the applicability of the two-state allosteric model will depend on the observations to
which it is applied and the systems in which it is tested. The
allosteric two-state model would be most suitable, for instance, at ion
channel-linked receptors, where the production of stimulus is
equivalent to production of response. One interesting prediction of the
model is the property of coagonism, whereby an allosteric modulator can
modify orthosteric ligand intrinsic efficacy without itself possessing
any efficacy; this is embodied in the parameter, 
, in Fig. 2D.
Coagonism is commonly observed for ligands acting at the NMDA receptor,
for example (Corsi et al., 1996).
B. Behavior of the Ternary Complex Model
Allosteric interactions at GPCRs can be manifested in a variety of
ways. A useful means of obtaining a picture of the possible repertoire
of behaviors displayed by allosteric ligands is to simulate them using
one of the allosteric ternary complex models introduced above and to
compare the predications of the model with experimental observations.
When choosing the most appropriate model for such an exercise, a
trade-off needs to be made between number of model parameters and
parsimony in model predictive capabilities. For this reason, the simple
allosteric TCM (e.g., Fig. 2B) remains the most parsimonious and most
commonly used model for both prediction and quantification of
allosteric interactions at GPCRs (Ehlert, 1988; Lazareno and Birdsall,
1995; Christopoulos, 2000a,b, 2002). At best, the model can be used to
derive actual estimates of cooperativity factors and ligand affinities
under the appropriate experimental conditions. At worst, it can provide
semiquantitative or operational parameters that can still be useful in
system characterization and/or subsequent experimental design. Thus,
some discussion about the operational behavior of the simple allosteric
TCM is warranted.
As outlined previously, the simple allosteric TCM at GPCRs involves the
concomitant binding of two ligands, A and B, to the one receptor, R, to
form a ternary complex, ARB. For illustrative purposes, Scheme
1 will be adopted.
Ligand A binds to the orthosteric site, whereas ligand B, the
allosteric modulator, binds to the allosteric site. The constants Ka and
Kb denote the equilibrium association
constants for the binding of A and B, respectively, to their binding
sites on the unoccupied receptor. In this regard, each of these
bimolecular reactions is no different from the standard mass-action
schemes applied to orthosteric binding. However, allosteric
interactions are not only characterized by unconditional ligand
affinity constants, but also by the cooperativity factor denoted here
by the symbol . Values of > 1 denote positive
cooperativity, whereas < 1 denotes negative cooperativity.
Values of approaching zero would be indistinguishable from
competitive antagonism. In contrast, an value equal to 1 denotes an
allosteric interaction that results in unaltered ligand affinity at
equilibrium. Allosteric interactions can still be discerned under
nonequilibrium conditions, and this is discussed later (vide infra).
In addition to the well characterized allosteric effects between
agonists and G proteins occurring at GPCRs, a growing number of studies
are identifying additional allosteric sites located on specific GPCRs.
The best studied examples involve the muscarinic acetylcholine
receptors, with allosteric interactions having been conclusively
demonstrated at all five subtypes of these receptors (Henis et al.,
1989; Lee and El-Fakahany, 1991; Tucek and Proska, 1995; see Birdsall
et al., 1996; Ellis, 1997; Christopoulos et al., 1998; Holzgrabe and
Mohr, 1998). However, allosteric interactions between various ligands
have also been demonstrated at other GPCRs, as shown in Table
2. Although this may seem to be a rather
diverse list of receptors, allosteric interactions at GPCRs share a
number of common features that allow them to be detected and possibly used in a therapeutic sense.
From the simple scheme described above, fractional receptor occupancy
by the orthosteric ligand A (
A) is equal to
([AR] + [ARB]/[R]) and is expressed as
![&rgr;<SUB><UP>A</UP></SUB>=<FR><NU><FR><NU>[<UP>A</UP>]</NU><DE>K<SUB><UP>A</UP></SUB></DE></FR></NU><DE><FR><NU>[<UP>A</UP>]</NU><DE>K<SUB><UP>A</UP></SUB></DE></FR>+<FR><NU>(1+[<UP>B</UP>]/K<SUB><UP>B</UP></SUB>)</NU><DE>(1+&agr;[<UP>B</UP>]/K<SUB><UP>B</UP></SUB>)</DE></FR></DE></FR>](/content/vol54/issue2/fulltext/323/img005.gif)
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(5)
|
where KA and
KB denote the equilibrium dissociation
constants of A and B, respectively, at the free receptor. In the
absence of allosteric modulator, the receptor occupancy of the
orthosteric site is determined by the orthosteric ligand's equilibrium
dissociation constant, KA. However,
when an allosteric ligand is present, the occupancy of the orthosteric
site will now be determined by the following composite parameter,
KApp
![K<SUB><UP>App</UP></SUB>=<FR><NU>K<SUB><UP>A</UP></SUB>(1+[<UP>B</UP>]/K<SUB><UP>B</UP></SUB>)</NU><DE>(1+&agr;[<UP>B</UP>]/K<SUB><UP>B</UP></SUB>)</DE></FR>](/content/vol54/issue2/fulltext/323/img006.gif)
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(6)
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If the interaction between A and B is positively cooperative
( > 1), then KApp < KA and the binding curve of ligand A
at the modulator-occupied receptor will be shifted to the left relative to the binding curve of A at the free receptor. In contrast, negative cooperativity between A and B ( < 1) will be manifested as a rightward displacement of the binding curve for A (i.e.,
KApp > KA). Figure
4 illustrates these relationships for the
binding of an orthosteric ligand in the presence of increasing
concentrations of an allosteric modulator with an value of either
0.1 (negative cooperativity) or 10 (positive cooperativity). This
figure also illustrates an important aspect of allosteric interactions,
namely that these types of interactions approach a limit, the extent of
which is governed by the magnitude of . The closer the value of is to 1, the more readily the limit is approached with increasing concentrations of B.
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Fig. 4.
Effect of a negative allosteric modulator (A),
positive allosteric modulator (B), or a competitive antagonist (C) on
orthosteric ligand-receptor occupancy (A) based on the
simple ternary complex model for allosteric interactions (eq. 5). For
all the simulations, pKA = 6 and
pKB = 9. The modulator, B, modifies
orthosteric ligand affinity to a limit determined by the cooperativity
factor () that characterizes the interaction between allosteric and
orthosteric sites. In these examples, ligand affinity is either
maximally diminished (A) or enhanced (B) by a factor of 10. In
contrast, simple competitive interactions (C) are characterized by
mutually exclusive binding of the two ligands for the same site and,
thus, allow for a theoretically limitless dextral shift of orthosteric
ligand occupancy.
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C. The Molecular Nature of Allosterism at G Protein-Coupled
Receptors
The ability of orthosteric ligands, once bound, to modify the
signaling properties of receptors has been defined as a measure of
orthosteric ligand efficacy (Kenakin, 1996a, 2002). The very nature of
efficacy is intertwined with the ability of the orthosteric ligand to
produce a conformation of the receptor that either promotes signaling
(as is seen with agonists) or attenuates constitutive receptor
signaling (as is observed with inverse agonists). Because the binding
of an allosteric modulator to a distinct accessory site on the receptor
causes its own alteration of receptor conformation, it is conceivable
that the resulting conformation may influence orthosteric ligand
efficacy, in addition to the effects on orthosteric ligand affinity
described in the preceding section. Thus, although assays of receptor
signaling are necessarily influenced by post-binding stimulus-response
events, they nevertheless afford the opportunity to detect specific
receptor conformations promoted by allosteric modulators that may not
necessarily be evident in radioligand binding assays.
When considering the conformational space of GPCRs, it is often
parsimonious to confine GPCR activity to two states (an inactive state
that does not activate G proteins and an active state that does).
However, there are no data to suggest that agonists simply enrich a
single population of active receptor state to produce response. It is
well established that proteins exist in numerous conformations or
substates (Frauenfelder et al., 1988, 1991; Frauenfelder, 1995).
Thermal energy causes fluctuation between these states with certain
low-energy states being "favored" (Gerstein et al., 1994; Haltia
and Freire, 1995). Although the ETC and CTC models are sometimes
referred to as two-state models, this is a misnomer from the point of
view of ligand activation. The two states R and R* refer to the
unliganded forms of the receptor, and upon binding of ligand, the
factors and (and additionally for the CTC model) confer
complete ligand specificity to the protein species. Under these
circumstances, these models are infinite-state models because ligands
could have unique values for , , and (Watson et al., 2000).
This introduces the concept of G protein- and ligand-selective receptor
active states.
1. G Protein-Specific Receptor Conformations.
There are
numerous lines of evidence to suggest that different agonists produce
response through the formation of different receptor active states. The
most compelling data are obtained from receptors that are pleiotropic
with respect to the G proteins with which they interact because these
different G proteins provide a means of differentiating signaling
active states. From this standpoint, the pattern of activation of
various stimulus-response pathways can be used to infer the existence
of these states. This phenomenom is termed "stimulus trafficking",
whereby agonists differ in the ability to stimulate separate
stimulus-response pathways through a single receptor (Kenakin, 1995a,
1995b, 1997a).
It is known that different regions of the cytosolic loops of GPCRs
activate different G proteins (Ikezu et al., 1992; Wade et al., 1999),
and it would not be expected that different tertiary conformations of
the receptor protein would expose these different regions in an
identical manner. Therefore, if ligands produce different tertiary
conformations, then these may be detected through the relative
capabilities of the resulting species to activate different G proteins.
This should not be confused with differential activation of pathways
through strength of stimulus. If a receptor couples to one pathway with
great efficiency and to another one poorly, a strong agonist with high
efficacy may activate both pathways, whereas a weaker agonist would
activate only the most efficiently coupled pathway; this is not
stimulus trafficking. To conclude true differences in receptor active
state, a reversal of potency for the pathways or differences in the
maximal activation of the pathways by the agonists must be
demonstrated. This has been shown for some receptors. For example, the
human 5-HT2C receptor is coupled to two separate
response pathways in CHO cells, namely phospholipase
A2-mediated arachadonic acid release and
phospholipase C-mediated inositol phosphate accumulation (IP
accumulation). There is a striking reversal in the maximal responses of
agonists in this system that cannot be accommodated by postulating the production of a single receptor active state. Thus, the agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane produces a higher maximal stimulation than the 5-HT agonist quipazine for arachadonic acid release (Berg et al., 1998). Because efficacy is the sole receptor-related determinant of maximal response, these data indicate that (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane has a greater efficacy for IP accumulation than quipazine for arachadonic acid release. This relative efficacy for these agonists is reversed for IP
accumulation where quipazine has the greater efficacy. Thus, a
receptor-related parameter, namely efficacy, reverses with the two
agonists for the same receptor. Similarly, there is a reversal of the
relative potency of substance P analogs on neurokinin NK-1 receptors
described where substance P is 2.1 times more potent than the analog
[P3Emet(O2)11]SP
for producing cyclic AMP through NK-1 receptor activation, but is 0.11 times less potent than the analog for producing phosphoinositol hydrolysis through activation of the same receptor (Sagan et al., 1999). Reversals of efficacy also have been reported for pituitary adenylate cyclase-activating polypeptide receptors (Spengler et al.,
1993), dopamine receptors (Meller et al., 1992), and
Drosophila tyramine receptors (Robb et al., 1994). In
general, these data cannot accommodate a mechanism whereby all of the
agonists involved produce an identical active receptor state.
Specially designed recombinant GPCR systems (termed
"stimulus-biased" assay systems; Watson et al., 2000) also can be
used to detect stimulus trafficking. These systems consist of surrogate host cells for receptor transfection with identical cellular
backgrounds except for the enrichment of a single G-subunit. A study
with human calcitonin receptor (type 2), a pleiotropic receptor that can interact with Gs, Gq,
and Gi, (Horne et al., 1994), showed striking
reversals in relative potencies of peptide calcitonin agonists.
Specifically, after transfection of the receptors into wild-type HEK
293 cells and HEK cells stably transfected with enriched populations of
G-subunits, differences in relative agonist potencies were observed.
For example, the relative potency of porcine calcitonin and rat amylin
changed by a factor of 18 (from 4.6 to 84) when compared in wild-type
and Gs-enriched cells. This suggests that
porcine calcitonin produces a conformation more conducive to using
Gs than does amylin. In these studies, even the
rank order of potency of the agonists changed in that the potency of
rat calcitonin gene-related peptide (CGRP) was 0.3 times that of rat
amylin in wild-type cells and three times greater than rat amylin in
Gs-enriched host cells (Watson et al., 2000).
Because the classification of receptors using agonist potency ratios
and rank orders of potency is based on the tenet that the active state
produced by the agonists is the same, deviations from this behavior
suggest that the tenet is not valid in this system.
Another observation not consistent with the idea that agonists simply
enrich the spontaneously formed receptor active state is the phenomenom
of "protean agonism". This behavior has been described in
theoretical terms as the formation, by an agonist, of a receptor active
state that is less efficacious than the spontaneously formed
constitutive one (Kenakin, 1995c, 1997b). It was named for the Greek
god Proteus who could change shape at will. The hallmark of protean
agonists is the production of positive agonist response in
nonconstitutively active systems and inverse agonism in constitutively
active ones. Such a pattern of response can be used as presumptive
evidence that the agonist produces a receptor active state that is
different (i.e., of lower efficacy) than the spontaneously formed
active state, i.e., ligand selective agonism. Under these
circumstances, protean agonism can be considered a looking glass into
receptor states.
There are theoretical conditions under which protean agonism could
occur. For example, in the CTC, a ligand could promote the R* form of
the receptor by having > 1 but then produce a liganded form
of the receptor active state of lower affinity than the unliganded form
( < 1); the result would be a reversal of the positive to a
negative agonism under conditions of constitutive activity.
Importantly, there are also experimental examples of protean agonism.
The -adrenoceptor ligand dichloroisoproterenol has been shown to
produce positive partial agonism in Sf9 cells transfected with
2-adrenoceptors. When membranes were prepared from these same cells, the system demonstrated constitutive activity (due to removal of cellular GTP) and dichloroisoproterenol then became
an inverse agonist. The same behavior was observed for the ligands
labetolol and pindolol (Chidiac et al., 1994, 1996).
The kinetics of cyclic AMP formation have been used to detect
agonist-selective receptor states. Thus, in the presence of limiting
GTP concentrations, such kinetics indicate a differential rate of
heterotrimer dissociation of G protein subunits with different -adrenoceptor agonists (Krumins and Barber, 1997). Similarly, differences in the ability of -adrenoceptor agonists to hydrolyze inosine versus guanosine triphosphate suggest the formation of ligand-specific receptor active states as well (Seifert et al., 1999).
Mutation studies also suggest that ligands stabilize different tertiary
conformations of receptors. For example, mutations of dopamine
D2 receptors produce agonist-specific abolition
of G protein activation (Wiens et al., 1998). Desensitization of receptors by some agonists also suggests differential receptor active
state formation. Whereas it would be expected that the ability of
agonists to induce desensitization would parallel their ability to
produce response (i.e., intrinsic efficacy), studies on µ-opioid
receptors have indicated a disproportionate desensitizing and receptor
phosphorylating property of methadone and L--acetyl methadone, thereby, suggesting different receptor conformational changes with these ligands (Yu et al., 1997). Differential
desensitization also has been demonstrated for methadone and
buprenorphine on µ-opioid receptors (Blake et al., 1997).
Studies with purified -adrenoceptor covalently labeled with
cysteines with an environmentally sensitive fluorophore
4[(iodoacetoxy)ethylemethylamino]-7-nitro-2,1,3-benzoxadiazole allowed observation of changes in protein conformation with ligand binding (Gether et al., 1995). A statistical analysis of these data
indicates serious deviation from a simple two-state model of receptor
activation suggesting that different ligands produce uniquely different
protein conformations (Onaran et al., 2000).
The major window of detection of allosteric effects
historically has been receptor-mediated physiological response. Thus, ligands have been detected as allosteric modulators or enhancers on the
basis of effects resulting in changes in tracer ligand affinity and/or
tracer ligand-induced response. However, different receptor
conformations are involved in receptor-mediated effects other than
cellular signaling (Kenakin, 2002). Thus, conformations resulting in
changes in receptor phosphorylation and/or receptor internalization
also can be relevant to the therapeutic effect of allosteric ligands.
For example, studies on receptor internalization suggest
ligand-specific receptor conformations. Thus, the cholecystokinin receptor antagonist D-Tyr-Gly-[(Nle
28,31,D-Trp30)cholecystokinin-26-32]-phenethyl
ester is an antagonist on the receptor producing blockade of responses
to cholecystokinin but produces profound acceleration of receptor
internalization (Roettger et al., 1997). This indicates the formation
of a unique conformation that does not signal to G proteins but is more
amenable to receptor phosphorylation and subsequent internalization.
Similarly, whereas enkephalins and morphine both stimulate - and
µ-opioid receptors, enkephalins induce rapid receptor internalization
while morphine does not (Keith et al., 1996).
2. Ligand-Specific Receptor Conformations.
Although the
preceding discussion of specific receptor conformations focused on the
receptor-G protein interaction, it is evident that the entire surface
of a GPCR may be viewed as a potential binding site, and any ligand
binding to either the orthosteric or allosteric site(s) on a GPCR has
the potential to alter receptor conformation such that the affinity
and/or intrinsic efficacy of a ligand binding to the other site(s) on
the GPCR will also change. This scheme is also compatible with the
potential for multiple ligand-specific receptor conformations to be
engendered depending on the binding site and extent of conformational
change induced in the receptor protein. Thus, ligands that would be
classed as allosteric modulators with respect to their effects on the endogenous orthosteric agonist for the receptor of interest should be
placed in the same realm as other modifiers of receptor properties, such as agonists, inverse agonists, and G proteins. At the molecular level, therefore, the classic TCM of allosteric interactions and its
variants (Fig. 2) are all subsets of a more general, extended, model of
receptor activity. To visualize such a model, one can begin with a
general picture of a receptor protein that contains separate binding
sites for an orthosteric ligand, an allosteric modulator, and a G
protein. Thermodynamic considerations imply that the occupancy of any
one of the binding sites on this receptor can alter its conformation
such that the occupancy of any of the other sites on the protein is
also altered. This cross-reciprocity can be quantified in terms of
separate cooperativity factors for the interaction between orthosteric
and allosteric sites, orthosteric and G protein sites, and allosteric
and G protein sites. Because efficacy at GPCRs is invariably related to
the ability of the receptor to interact with its cognate G protein(s),
then efficacy at the molecular level can be impacted not only by the
interaction between orthosteric ligand and G protein or orthosteric
ligand and allosteric modulator (e.g., Section IIB), but also by the interaction of the allosteric modulator and the G protein. For instance, Fig. 5A shows the effects of
the allosteric modulator alcuronium on PI hydrolysis in CHO cells
transfected with the human M1 muscarinic
acetylcholine receptor (Jakubík et al., 1996). Even in the
absence of the muscarinic agonist carbachol, alcuronium was able to
elicit a significant stimulatory effect on PI hydrolysis that was
insensitive to antagonism of the orthosteric site by the classical
muscarinic antagonist quinuclidinyl benzilate. The effect of alcuronium
on PI hydrolysis was absent in cells that did not express the
M1 muscarinic receptor. Thus, it can be concluded that alcuronium was promoting receptor-G protein coupling via an action
at the allosteric site on M1 receptors. In a
similar manner, the allosteric modulator gallamine was also found to
activate the M1, M2, and
M4 muscarinic receptors in the absence of any other ligand (Jakubík et al., 1996), although it inhibits the binding of the endogenous muscarinic agonist acetylcholine at the same
receptors (Lazareno and Birdsall, 1995). This latter finding is a
striking example of ligand-specific receptor conformations, whereby
gallamine (and alcuronium) can promote conformations that
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