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Department of Receptor Biochemistry, Glaxo SmithKline Research and Development, Research Triangle Park, North Carolina 27709, USA
1Correspondence: Department of Receptor Biochemistry, GlaxoWellcome Research and Development, 5 Moore Dr., Research Triangle Park, NC 27709, USA. E-mail: tpk1348{at}glaxo.com
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ABSTRACT |
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 TOP ABSTRACT NEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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Key Words: G-protein-coupled receptors • receptor activation • inverse agonism • receptor theory • constitutive receptor activity
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NEW MOLECULAR TARGETS FOR DRUG DISCOVERY |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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). Before 1995, the major targets for drug development were full and
partial agonists and antagonists. Since the principal mode of
high-throughput screening has been radioligand binding, orthosteric
ligands (those that sterically hinder the access of the radiolabel to
the receptor binding site) primarily were discovered. Allosteric
ligands (those that affect receptor function through binding to their
own binding site separate from that of the endogenous ligand) were
detected only if the allosteric interaction resulted in an alteration
of the affinity of the receptor for the radiolabel. With the
technological advances enabling high-throughput functional
receptor screens should come an increase in the types of GPCR ligands
in the new millennium. This will result in an increase in the number of
allosteric ligands (modulators, agonists, enhancers) that modify
receptor function without necessarily modifying steric access of the
endogenous ligands to the receptor. Therefore, the changing mode of
high-throughput screening can be predicted to lead to an increase in
the texture of drug types for GPCRs. Another reason for the increasing
number of drug targets is increased knowledge of GPCR behavior in
cellular systems. This has led to the discovery of inverse agonism.
This review will concentrate on a subset of these new chemical targets,
namely, those that possess efficacy, either positive or negative, and
these will be discussed in terms of their mechanisms of action and
possible relevance to therapy of disease.
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Drugs with ‘efficacy’
Drugs can be thought of as having two properties with respect to
biological systems: affinity for the receptor and intrinsic efficacy. A
common usage of the word efficacy in clinical pharmacology is
‘therapeutically useful activity’. Thus, a drug is considered
‘efficacious’ if it alleviates the symptoms of a disease in a
patient. Within this context, even a competitive antagonist would have
‘efficacy’. This review will discuss efficacy in terms of its formal
definition in pharmacological receptor theory, that is, the property of
a molecule that causes it to produce some observable physiological
response. In terms of GPCRs, a useful working definition of receptor is
the property of a molecule that causes the receptor to change its
behavior toward the host system (1)
.
Three types of efficacious drugs will be discussed. Inverse agonists are an established drug class and possess what is termed ‘negative efficacy’. Protean agonists are a theoretical class that produce receptor activation of lower magnitude than that emanating from spontaneous receptor constitutive activity. The predicted behavior for this class would be the observation of positive agonism in some GPCR systems and inverse agonism in others. Although this has been observed experimentally, an explanation of the effect in terms of receptor conformation is still theoretical. Finally, ‘ligand-specific’ agonism, which considers that some agonists have a different quality as well as quantity of efficacy for a given GPCR, will be considered. All of these classes will be discussed in terms of the evidence for their classification and their possible therapeutic relevance. As a preface to discussion of these drug entities, it is useful to discuss the dynamics of the GPCR systems with which they interact.
GPCR systems
G-protein-coupled receptors are allosteric proteins designed by
nature to respond to small ‘drug-like’ molecules (i.e.,
neurotransmitters) to affect changes in large protein–protein
interaction (receptors and G-proteins). The common currency of this
translation of information is receptor protein conformation. It is
essential to understand three particular properties of GPCR systems in
order to understand how ligands can function as inverse, protean, and
structure-specific agonists. The first is that, like all proteins,
receptors can exist in various conformations. However, in the case of
GPCRs, some of these conformations reveal sequences in their cytosolic
loops, which can then activate G-proteins to initiate response. These
conformations are referred to as the ‘active state’ (Ra) of the
receptor; correspondingly, the conformation(s) that do not activate
G-proteins are referred to as the ‘inactive state’ (Ri). In the
simplest case, one single conformation of each will be assumed with the
two conformations existing in an equilibrium defined by an ‘allosteric
constant’ (denoted L and defined as [Ra]/[Ri]).
A second property of GPCR systems is that they are synoptic and
interactive. Therefore, it is incorrect to describe GPCR function
simply in terms of the receptor (two-state theory). Rather, the
G-protein is an interactive and essential part of the system. The
G-protein influences the receptor in ways that modify the behavior of
the receptor and vice versa. Of particular relevance is the fact that a
receptor can spontaneously interact with G-proteins in the absence of
agonist ligands. Thus, if the affinity of Ri for a G-protein is denoted
Kg (equilibrium association constant), the affinity of the active state
Ra for the same protein is denoted ßKg where ß>1. Response
emanates from the hydrolysis of GTP by the G-protein resulting from
activation by Ra. From these elements the simplest version of a GPCR
system can be constructed:
 |
 |
 |
, 3)
or point
mutation (4
5
6
7
8
9
10)
can affect L and make receptors more
constitutively active.
The simple model for GPCR systems can be completed by adding the
interaction of ligand (designated [A]) to the system to produce a
corresponding array of species ARi, ARa, and ARaG. When the
ligand-bound ensemble is added to the scheme shown in Equation 1
(see
also Fig. 2A
), the extended ternary complex model (ETC model) for GPCR
systems results (5)
. A more thermodynamically complete
version of the system allows the inactive receptor Ri to interact with
the G-protein. In terms of thermodynamic modeling, this must be allowed
to occur (11)
. However, the existence of an inactive
ternary complex comprising ARiG is largely theoretical. Some examples
of this complex can be found for some receptors (see ref
12
for a review); however, the thermodynamically complete
model for GPCR systems, termed the cubic ternary complex model (CTC
model; 13
14
15
), requires a greater number of microaffinity
constants than the ETC model and generally is more complex (see Fig. 2B
). The ETC model can be regarded as a subset of the CTC
model and adequate for GPCR systems for which the interaction of Ri
with G-protein is thought to be minimal. For the purposes of this
review, both models yield similar predictions for GPCR behavior with
some minor exceptions.
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The third relevant property of GPCR systems is an extension of the
first: the production of multiple active receptor states
(that go on to produce response through interaction with G-proteins).
The minimal requirement for a GPCR model is that one receptor active
state be formed. Thus, in principle, agonists can induce response by
causing enrichment of that single receptor active state. Under these
circumstances, efficacy would then be a matter of the quantity of the
active state produced by the agonist. However, there is no theoretical
constraint on the number of receptor active states. Even though the ETC
and CTC model have both been referred to as ‘two-state’ models, this
is a misnomer in that there is the capability within both to be
multi-state models. The two-state aspect of these models refers only to
the unliganded species Ri and Ra. In principle, the microaffinity
constant of the liganded receptor could be specific for the ligand
(through the values 
and 
for the ETC model and , , and 
for the CTC model; see Fig. 2
), i.e., the affinity of the ligand-bound
receptor for G-protein (ARaG) could be different from the unbound form
(RaG). Under these circumstances, both the ETC and CTC models can
accommodate an infinite number of receptor active states for agonism.
It is clear that proteins, including GPCRs, can adopt numerous
conformations according to thermal energy (16
, 17)
. What
is not clear is what proportion of these conformations are capable of
activating G-proteins, i.e., how many are receptor active states? Amino
acid sequences have been identified in the intracellular loops of GPCRs
that, when exposed to G-proteins, activate them (18
19
20)
.
In fact, small oligopeptide isolated sequences have been found to
activate G-proteins on their own (21
, 22)
. With this model
in mind, it would suggest that the inactive form of the receptor
prevents access of G-proteins to these sequences, thereby precluding
receptor activation of G-proteins. The corollary to this is that any
disruption of the tertiary structure of the receptor could expose these
activating amino acid sequences to initiate G-protein activation. On
theoretical grounds, it might be expected that there could be numerous
tertiary conformations of the receptor capable of exposing these
intracellular sequences, i.e., there could be numerous active state
conformations of the receptor. Mutation studies support this idea. For
example, the substitution of 20 amino acids in position 293 of the
1A-adrenoreceptor produces a constitutively
active receptor—essentially 20 different active state similar forms of
the 1A-adrenoreceptor (4)
. The
production of constitutive activity (whereby the receptor spontaneously
adopts an active state and produces G-protein activation) through such
mutations for receptors indicates that disruption of receptor tertiary
conformation can expose activating sequences to G-proteins (10
, 23
, 24)
. A general message from these studies is the possibility
of the existence of numerous active state conformations of GPCR able to
initiate physiological response. The apparent ligand-specific
production of receptor conformations that interact differently toward
other membrane proteins (including G-proteins), to be discussed later
in the context of ligand-specific agonist efficacy, further suggest the
existence of multiple receptor active states for GPCRs.
The previous discussion has described essentially three characteristic behaviors of GPCR systems: the capability to exist in multiple states, the ability of these states to spontaneously interact with other membrane proteins, and the possible existence of multiple states capable of inducing physiological response. To explore the interaction of ligands with such systems, it is useful first to discuss the mechanism by which ligands can influence receptor/G-protein ensembles.
The influence of ligands on GPCR systems
The relative quantities of various protein species existing in
equilibria with each other are governed by the equilibrium dissociation
constants that define their ratio. Thus, the allosteric constant is
defined as [Ra]/[Ri]. The nature of L is controlled by the
molecular nature of the receptor; thus, for any quantity of Ri there
will be a quantity of Ra governed by the magnitude of L. However, this
can be changed if external forces perturb the quantity of either one of
the species. For example, if a ligand binds selectively to the Ra
species to form ARa, then the quantity of free Ra is depleted and the
magnitude of L will dictate that more Ra must be formed at the expense
of existing Ri (see Scheme 21).
This can be shown mathematically within the constraints of either the
ETC or CTC model (Fig. 2)
. For example, the concentration of response
producing species (RaG and ARaG) in the presence of a ligand A in terms
of the ETC model is given by Kenakin et al. (12)
:
 |
 |
)
 |
 |
, and reflect modifiers of the
affinity constant of the receptor for the G-protein when the receptor
is activated and occupied by ligand, respectively. For example, a value
of > 1 indicates a greater affinity of the ligand for the
active-state receptor Ra. It can be seen from Equation 7
that only one
condition will enable a ligand to bind to the GPCR species in the
system and not cause a redistribution of receptor species.
That is if = = 1 (the presence of the ligand on the
receptor does not in any way affect the affinity of the receptor for
G-proteins, i.e., the ligand has no efficacy). If or 
1, then the ratio of active-state species will change in the presence
of A: when A is added to the system, the concentrations of the various
species will redistribute. Therefore, the selective affinity of ligands
for various receptor conformations will change the overall distribution
of species in GPCR receptor ensembles and thus, either induce or
inhibit response. This is the basic mechanism of ligand efficacy and
the basis for the molecular nature of inverse, protean, and
ligand-selective agonism. |
INVERSE AGONISTS |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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, 26)
, true GPCR constitutive activity was first quantified in
recombinant receptor systems where experimental conditions could be
manipulated to produce constitutive activity. As noted, the ability to
spontaneously produce an active receptor conformation (the conformation
that activates G-proteins) is defined by the allosteric constant, a
receptor-specific constant defining the energy barriers to the
production of tertiary conformations. Thus, in natural systems with
defined receptor/G-protein stoichiometry, the amount of spontaneously
formed active-state receptor species may not be sufficient to
demonstrate visible constitutive receptor activity. This constraint was
eliminated by the introduction of recombinant GPCR systems, which could
experimentally manipulate the relative stoichiometry of receptors and
G-proteins.
A classic study by Costa and Herz (2)
of NG108–15 cells
recombinantly expressing opioid receptors was instrumental in defining
constitutive GPCR activity and inverse agonism. Costa and Herz
(2)
produced a system that responded to the classic opioid
agonist (i.e., [D-Ala2), D-Leu]enkephalin), but
also had an elevated basal response and demonstrated a depression of
basal activity with the peptide ICI 174864
([N,N'-diallyl-Tyr1,Aib2,3]Leu5-enkephalin).
In this constitutively active GPCR system, ICI 174864 depressed the
ligand-independent elevated basal responses and was thus defined as an
inverse agonist. The simplest mechanism by which inverse agonism could
occur is the selective affinity of the ligand for the inactive state of
the receptor. Thus, as the ligand binds selectively to Ri, the receptor
species in the system will redistribute. If the system has RaG present
(constitutive activity), then this species will be depleted as more
receptor transforms into ligand-bound Ri; the result will be a decrease
in constitutive activity.
Inverse agonism is a fairly newly discovered phenomenon for GPCR
systems. The effect was initially met with some skepticism since it
required the reclassification of established antagonists as inverse
agonists. Also, in some systems the trace presence of endogenous
agonists leads to an apparent constitutive activity, which could then
be depressed by simple competitive antagonists, i.e., inverse agonism
could be an artifact in some systems. However, the lack of depression
of basal responses to some antagonists (i.e., neutral antagonists) and
the use of such neutral antagonists to block the effects of inverse
agonists clearly indicate that the phenomenon is real. For example,
Costa and Herz (2)
used the neutral antagonist MR 2266 to
block the effects of the positive agonist DADLE and the inverse agonist
ICI 174864, and showed that the potency for the inhibition of both
effects was the same.
After the initial discovery, there was a period when there was a paucity of data available to judge the prevalence of inverse agonists in pharmacology. However, with time has come an increasing number of reports describing previously classified antagonists as inverse agonists. This rise coincided with the increased availability of recombinant and constitutively active GPCR systems, a prerequisite for the observation of inverse agonism. Thus, now that more laboratories have eyes to see inverse agonism, the more it has been seen.
It is still premature to judge the prevalence of inverse agonism in
chemical space. In theoretical terms, there is reason to believe that
all ligands should not possess efficacy. As described above, for a
ligand not to cause redistribution of GPCR species it must
recognize at least two receptor conformational species as being
identical: Ra and Ri. In a constitutively active system, this is
increased to three species by the presence of RaG. As shown in Equation 7
, the ligand-specific constants and must be unity in terms of
the ETC model (and , , in the CTC model) for redistribution
not to occur (i.e., for a ligand to have no efficacy). The question
then is: How often, in thermodynamic terms, is this likely to occur?
Although some studies appear to support the prediction that most
antagonists are inverse agonists (i.e., of 23
1-adrenoreceptor antagonists of varying
structure, all were inverse agonists) (27)
, there are
clear examples of neutral antagonists in the literature. The degree of
inverse agonism observed depends on the relative affinity of the
inverse agonist for the various receptor species and the degree of
constitutive activity in the system. Thus, ligands that only slightly
differentiate receptor conformations will essentially appear to be
neutral antagonists, especially in systems with low levels of
constitutive activity.
Although the existence of inverse agonists has been substantiated in experimental systems, the therapeutic relevance of this drug class is as yet unknown. It also is not clear whether negative efficacy would be a desirable or undesirable property to have in an antagonist molecule. In the absence of constitutive receptor activity, an inverse agonist behaves exactly as a simple competitive antagonist. However, if there is constitutive activity present in the therapeutic system, then, unlike a simple competitive antagonist, an inverse agonist will depress the resulting elevated basal response. There are physiological scenarios where this may or may not be advantageous.
Adverse effects of inverse agonists
Inverse agonism has been associated with receptor up-regulation
leading to tolerance to chronic antagonism. For example, in treating an
ulcer, tolerance to some histamine H2 receptor antagonists has been
observed (28
29
30)
. It has been postulated that chronic
treatment with histamine antagonists results in increased levels of
membrane histamine receptors (31)
. The ligands shown to
cause increases in histamine H2 receptor density—cimetidine and
ranitidine—are inverse agonists but there is no concomitant increase
in receptor density observed with the neutral antagonist burimamide
(32)
. In that membrane receptor populations are not
static, but rather are a series of steady states resulting from
receptor synthesis, transport to the surface, internalization, and
degradation, any ligand that perturbs receptor states theoretically can
affect the steady-state level of the receptor density. For example,
activation by agonists increases phosphorylation of many receptors and
subsequent internalization (33
34
35)
. It has been shown
that spontaneous formation of receptor active states (constitutive
activity) leads to eventual internalization of receptor as well
(33
, 36)
. Possessing equal affinity for both the inactive
and active receptor states, a neutral agonist would not alter flow of
receptor to and from the membrane surface. However, an inverse agonist
could halt the spontaneous cycle of receptor synthesis, transport,
internalization, and degradation at the membrane by selectively
stabilizing the inactive state of the receptor. If this state is more
resistant to phosphorylation and subsequent internalization, then
receptor degradation would be slowed in the face of unaltered receptor
synthesis. The extent of change of steady-state membrane receptors
would be a function of the rates of the various processes synthesizing,
transporting, and internalizing them (37)
, but under
appropriate conditions elevations of receptor could occur leading to
increased agonist response. This, in turn, would result in a decrease
in the effectiveness of the antagonist. Thus, in this scenario, inverse
agonism would be an undesirable property (38)
. Receptor
up-regulation by inverse agonists has been shown to occur with inverse
agonists for histamine H2 receptors (32)
,
ß2-adrenoreceptors (39)
, and
1-adrenoreceptors (40)
. In
addition to changes in receptor density, inverse agonists have also
been found to alter levels of G-protein. Thus, up-regulation of levels
of Gq/11 through 5-HT2C
receptors (41)
and Gs through
ß2-adrenoreceptors (42)
has been
obtained with inverse agonists for the respective receptors. Presumably
the changes in receptor stimulation of these pathways leads to
secondary effects on G-proteins.
Therapeutic application of inverse agonists
The extent to which inverse agonism could be a therapeutic
advantage depends on the role of constitutive GPCR activity in
pathology. One potential therapeutic area where this might have
relevance is cancer. It has been shown that chronic elevation of second
messengers in cells produced by constitutive G-protein activity can
lead to cell transformation (43
44
45)
. For example,
receptors such as the 1-adrenoreceptor have
been shown to be agonist-independent proto-oncogenes (46)
.
Constitutive GPCR activity leading to chronic elevation of cell
metabolism may also have a role in promoting the growth of tumors.
There are examples of high levels of expression of specific GPCRs in
tumor cells; it has been shown that endogenous ligands for these
receptors are present at high levels in the tumor cells
(self-regulation) and that they have proliferative properties. There
also is evidence to show that inhibition of the cellular effects of
these ligands can inhibit tumor growth.
One such receptor is vasoactive intestinal peptide receptor (VIP).
Receptors for VIP are found in high density in a number of tumors
(47
48
49
50
51
52
53
54
55)
; see Table 1
. In fact, these high levels of VIP receptors can be used to image
tumors through binding of 123I-VIP
(55)
and 123I-labeled octreotide
(VIP ligand; 57
, 58
) binding.
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The relevance of high levels of VIP GPCR activity on tumors relates to
the fact that this peptide promotes growth and proliferation of normal
and malignant cells (59
60
61
62
63)
. Inhibition of VIP function
in these cells leads to a decrease in cancer growth (64
, 65)
; see Fig. 3
. The relevant question for inverse agonism is, to what extent can the
VIP-mediated proliferation be attributed to constitutive VIP GPCR
activity? Many of these tumors have high levels of VIP, and it has been
suggested that VIP secretion from these tumors regulates VIP receptor
expression on the same cells (66)
. Certainly the high
levels of VIP receptor present on the tumor cell membrane would make
them extremely sensitive to low levels of released VIP. However, the
sheer magnitude of the receptor expression suggests that constitutive
receptor activity may also play a role in the pathology.
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There are differences in the proclivity with which different GPCRs
spontaneously produce an active-state receptor (with corresponding
constitutive activity). Some receptors have a low-energy barrier for
the formation of Ra (i.e., human calcitonin, chemokine CCR5,
neuropeptide Y types 2 and 4) whereas others, such as NPY1, do not
readily produce constitutive activity (67)
; the difference
lies in the magnitude of the allosteric constant, L. However, since the
definition of L is the ratio of Ra to Ri (L=[Ra]/[Ri]), then
irrespective of the magnitude of L, a 1000- to 10,000-fold increase in
the number of receptors will lead to a corresponding 1000- to
10,000-fold increase in the number of spontaneously existing
active-state receptors. Thus, the magnitude of L for VIP would need to
be exceedingly small to prevent such high levels of receptor from
producing constitutive activity.
Another peptide of interest in cancer is bombesin. Bombesin,
gastrin-releasing peptide, and VIP are related in that VIP may induce
the release of bombesin/GRP in small cell lung cancer (65
, 68)
. Bombesin-like peptides are potent mitogens, and a role has
been proposed for them in oncogenesis and/or proliferation of malignant
cells (69)
. Bombesin/gastrin-releasing peptides are found
in high levels in small cell lung carcinomas, suggesting that these
could be autocrine factors for cancer growth (70
71
72)
. As
with VIP, blockade of bombesin activity through monoclonal antibodies
attenuates cancer growth (69)
.
Inverse agonists would both block the effects of humoral activation of these receptors on cancer cells (i.e., secreted VIP, bombesin) and constitutive activity in the tumor due to either receptor over-expression and/or mutation. Whereas the effect would be cytostatic rather than a cytocidal (tumor death would not be achieved), a reduction in tumor cell metabolic activity could be a useful adjunct to chemotherapy.
Certain disease states may be treated effectively only with
inverse agonists. These are instances where the pathological entity is
a constitutively active GPCR, which produces physiological response in
the absence of endogenous agonists. For example, certain pathological
mutations lead to constitutively active GPCRs, which in turn result in
diseases such as retinitis pigmentosa and hyperthyroidism (see review
by Spiegel, ref 73
). Constitutively active GPCRs may also
be important in autoimmune diseases (see review by de Ligt et al., ref
74
). Viral infection also can lead to constitutively
active GPCR pathology. For example, infection with Kaposi’s
sarcoma-associated herpes virus leads to expression of a constitutive
chemokine receptor, which in turn elevates IP3 to lead to cell
proliferation and continued viral replication (75
, 76)
.
In general, it still is not clear to what extent GPCR constitutive
activity plays a role in pathology. However, it is known that receptors
and enzymes levels change in conditions of trauma (hypoxia, ischemia,
physical damage), disease (inflammation, viral or bacterial infection),
or development (78
, 79)
. Although, in general, solid
examples of constitutive receptor activity playing a role in disease
are sparse, with the classification of clinically used inverse
agonists, the relationship between negative efficacy and therapeutic
utility should become clearer. Along with clarification of the role of
constitutively active GPCRs in pathophysiology will come a measure of
the value of inverse agonists in therapy.
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PROTEAN AGONISTS |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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, 81)
. These kinds of ligands were given the
name protean agonists after Proteus, the Greek god who could change
shape and appearance at will. In this case, the reversal from positive
to negative agonism is protean. If a given agonist produces a receptor
active state that is less efficacious (to be denoted [Ra']) than the
spontaneously formed one (denoted [Ra]), then in systems that are
quiescent (no constitutive activity), the ligand would produce
excitation by virtue of changing the predominant Ri into Ra'. However,
if the system were constitutively active (significant amount of Ra),
then the ligand would reduce the activity by changing Ra to Ra'.
Therefore, in quiescent systems the ligand would be a positive agonist
and in constitutively active systems it would be an inverse agonist.
Presently it is not clear what therapeutic relevance such an agonist
would have except perhaps to set the level of stimulation of a given
system to a constant level. Thus, if pathology produced constitutive
activity to create an overstimulation of the system or if the
endogenous stimulus to the system were to be diminished by pathology,
then a protean agonist would be useful if the maximal effect of the
agonist was appropriate. On the other hand, there is a considerable theoretical interest in protean ligands since they can act as a looking glass into agonist-specific receptor active states. Thus, the observation of protean agonism would be presumptive evidence that the ligand in question produces a receptor active state of lower intrinsic efficacy than the naturally occurring constitutively active state. It is worth considering the experimental conditions under which such protean agonism would be observed.
The starting point is to have a ligand that produces a positive agonist
response in a quiescent (nonconstitutively active) receptor system. It
might be supposed that the agonism should be partial (in keeping with a
less efficacious ligand-bound active state). However, saturation of
system stimulus-response mechanisms might allow low efficacy agonists
to produce the full system response; therefore, partial agonism may not
be a prerequisite. The next step is to observe the effect of ligand in
a system where the receptor is made to spontaneously form the natural
active state. For example, Fig. 4A
shows the effect of increasing the magnitude of the
allosteric constant L (as might be produced by removal of sodium ions)
in a hypothetical GPCR system. The ligand is a theoretical drug that
promotes the formation of the natural active state (=100) but forms
a ligand bound species that has a lower affinity for the G-protein than
the natural active state (ARa has a lower affinity for G than does Ra;
=0.01). Calculations with the CTC model show that in the quiescent
system (L=0.01), the ligand is a positive agonist. Changing L from 0.01
to 0.3 elevates the basal response of the system and causes the ligand
to demonstrate inverse agonism. Another way to produce constitutive
activity is by increasing the amount of G-protein available to interact
with the receptor (Equation 3)
. Under these circumstances, a similar
ligand (=0.03) will demonstrate protean agonism as well (Fig. 4B
). Another condition that may yield protean agonism is
when the receptor reactivity to the G-protein changes. For example,
Fig. 4C
shows that if the affinity of both Ri and Ra is
reduced for the G-protein (KG increases), as
might be produced by desensitization, an inversion of agonism for the
same ligand would be observed. Note how in this case the basal activity
is not altered.
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There have been experimentally observed instances of protean agonism
for ß2-adrenoreceptor ligands. For example,
dichloroisoproterenol (DCI) is a positive partial agonist for
ß2-adrenoreceptors transfected into sf9 cells.
Upon desensitization of the system through prolonged treatment with the
full agonist isoproterenol (as depicted in the simulation Fig. 4C
), DCI produces inverse agonism (82)
.
Figure 5
shows the effects of three ß2-adrenoreceptor
ligands on transfected sf9 whole cells; DCI, labetalol, and pindolol
all produce increases in cyclic AMP (positive agonism). However, when
membranes were made from the same cells, the system became
constitutively active (due to removal of GTP) and, under these
circumstances, these same ligands produced depression of basal cyclic
AMP levels (inverse agonism) (83)
. It is not clear to what
extent low efficacy receptor conformations are responsible for the
experimentally observed protean agonism. However, observation of the
phenomenon is suggestive of selective receptor states and this may be a
useful tool for discovery of ligand-specific receptor active-states.
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LIGAND-SPECIFIC RECEPTOR ACTIVE STATES |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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. Since maximal response is
dependent only on efficacy, this indicates that DOI has a greater
efficacy than quipazine for AA release. In contrast, the efficacies of
the two agonists are reversed for the IP accumulation where quipazine
has the greater efficacy. This cannot be explained by a uniform
active-state receptor interacting with the two pathways identically for
the two agonists, but rather it suggests that the active state formed
by DOI (arachidonic release-preferring) is different from that produced
by quipazine (IP accumulation-preferring). Similar reversals of
efficacy have been reported for PACAP (pituitary adenylate
cyclase-activating polypeptide) receptors (84)
, dopamine
D2 receptors (85)
and
Drosophila tyramine receptors (86)
. A striking
reversal of relative potency of substance P analogs on neurokinin-1
receptors has been reported (87)
. Thus, whereas substance
P is 2.1x more potent than the analog
[P3Emet(O2)
(11)
SP for producing cyclic AMP through NK-1 receptor
activation, it is 0.11x less potent than the analog for
producing phosphoinositol hydrolysis through activation of the same
receptor. Whereas no reversals in relative efficacy for agonists was
found in a study of CB1 cannabinoid receptors (known to activate both
Gs and Gi protein), marked discontinuities in the activity of agonists
were observed indicating that some agonists produced conformations that
favored one of the two G-proteins while others did not
(88)
.
Differential activation of G-proteins by receptors (referred to as
stimulus trafficking; refs 89
90
91
) cannot be accommodated
by a mechanism whereby one single receptor active state produced by all
agonists interacts with G-proteins. Although differential stimulus
pathway activation can occur through strength of signal type of
mechanism (i.e., a highly efficacious agonist may activate two pathways
whereas a weaker agonist may activate only the more sensitive one),
reversal of relative activity cannot be explained in this manner.
Rather, the two G-proteins involved must see different conformations.
It would be expected that different conformations of the receptor would
have differential activation reactivities to different G-proteins since
it is known that different areas of the cytosolic loops on receptors
activate different G-proteins (92
, 93)
. It would not be
expected that different tertiary conformations of the receptor would
expose these different G-protein-activating sequences in an identical
manner.
Stimulus trafficking can be detected in specially designed recombinant
GPCR systems. Referred to as stimulus-biased assay systems
(94)
, these are hosts with identical cellular backgrounds
except for the enrichment of a single G subunit. For example, human
calcitonin receptors are pleiotropic with respect to the G-proteins
with which they can interact (Gs, Gq, Gi; ref 95
).
Transfection of human calcitonin receptors (type 2, denoted hCTR2) into
wild-type HEK 293 cells and HEK cells stably transfected with enriched
populations of G subunits show striking differences in
relative agonist potencies. Figure 6A
shows that not only does the relative potency of eight
calcitonin agonists on hCTR2, transfected in wild-type cells, and HEK
cells stably enriched with Gs subunit change, but so does their rank
order of potency. Figure 6B, C
shows dose-response curves to
rat amylin and porcine calcitonin in wild-type cells and Gs-enriched
cells, respectively. It can be seen that the relative potency of the
agonists changes from 4.6 to 84 with Gs-enrichment, a finding that
cannot be accommodated by the assumption that both agonists produce the
same receptor active state (94)
. Rather ,it suggests that
porcine calcitonin produces a conformation more conducive to using Gs
than does amylin.
|
Other experimental approaches have furnished data to indicate
differential G-protein activation by different agonists produced by
agonist-specific receptor conformations. For example, the kinetics of
adenylate cyclase activity in the presence of limiting GTP
concentrations indicates a differential rate of heterotrimer
dissociation for different ß2-adrenoreceptor
agonists (96)
. Similarly, whereas the efficacy of
ß2-adrenoreceptor agonists for promoting GTP
hydrolysis correlates well for the efficacy of the agonists for
stimulating adenylate cyclase, the same is not true for the hydrolysis
of inosine triphosphate. The differences in the ability of different
agonists to hydrolyze GTP vs. ITP suggest that different receptor
active states are produced (97)
.
There are still other lines of evidence to suggest that agonists
produce ligand-specific receptor conformations. Selective mutations of
dopamine D2 receptors caused selective abolition
of receptor/G-protein activation by dopamine but not other dopamine
agonists. This suggests that these agonists produce different receptor
conformations interacting with G-protein (98)
. Studies of
the receptor desensitizing effects of different agonists also indicate
the production of ligand-specific receptor conformations. For example,
it would be expected that the relative propensity of agonists to induce
desensitization would parallel their relative efficacies. This was
shown to be generally true for µ opioid receptor agonists, with the
notable exception of methadone and L--acetyl methadone. These latter
agonists produced disproportionate desensitization and receptor
phosphorylation, suggesting different receptor conformational changes
(99)
. Similarly, methadone and buprenorphine have been
shown to demonstrate different desensitizing properties from morphine
on µ opioid receptors (100)
. In other studies of
recovery from desensitization, it has been shown that agonists appear
to produce different conformations. Thus, whereas the recovery from
prolonged activation of 5-HT3 receptor with
partial agonists is mono-exponential, it is sigmoidal (indicating 3
steps and 4 states) with full agonists (101)
.
The effects of agonists on receptor internalization also have furnished
interesting data regarding ligand-specific receptor conformation. Here
it can clearly be shown that the simple strength of receptor
stimulation can be differentiated from the ability of ligands to induce
receptor internalization. For example, the cholecystokinin (CCK)
receptor antagonist
D-Tyr-Gly-[(Nle28,31,D-Trp30)cholecystokinin-26–32]-phenethylester
does not produce receptor stimulation but rather blocks CCK responses.
This antagonist also produces profound receptor internalization
(102)
. Similarly, whereas enkephalins and morphine produce
stimulation of and µ opioid receptors, enkephalins induce rapid
receptor internalization whereas morphine does not (103)
.
These data indicate that the conformations that lead to response are
not necessarily the same as those that induce receptor internalization.
It also suggests that different agonists produce receptor conformations
with differential propensity to internalize.
In conclusion, diverse experimental approaches have provided evidence that ligands can stabilize different receptor conformations. Some of these conformations relate to receptor signaling, whereas others may relate to receptor sensitivity to endogenous agonist or presence on the cell membrane. The challenge is to exploit this behavior for therapeutic advantage.
Ligand-selective conformations and therapeutic utility: the quality
of efficacy
Historically, receptors have been thought of operationally in
terms of ‘on-off’ switches. In this context, efficacy was considered
to be the ‘on’ position and the only gradation available in this
scheme was degree of strength. With the possibility of
agonist-selective activation of receptors and the definition of
efficacy as a change in the behavior of receptors to their hosts comes
the capacity to control the ‘quality’ of efficacy as well.
In terms of signaling, a common quest in drug discovery is to obtain
ligands with a subset of activity for a given endogenous ligand
receptor system. Historically, the method for doing this was through
discovery of receptor subtypes. Thus, whereas epinephrine has a
plethora of metabolic activities in the body mediated by
ß-adrenoreceptors, selective agonist stimulation of only the
ß2-adrenoreceptor subtype provides useful
therapy for asthma. Stimulation of the receptor subtype reduces the
spectrum of metabolic responses produced by the general receptor
family. If it is accepted that different receptor conformations most
likely reveal different portions of the intracellular cytosolic loops
of GPCRs, then ligand selective receptor conformations can lead to
further selective directing of activation to G-proteins (trafficking of
receptor stimulus). Such trafficking has been shown in natural and
recombinant systems. For receptors that produce pleiotropic activation
of multiple G-proteins, this would limit the signaling pathway
activated by the particular ligand and thus confer further selectivity
to the agonist (see Fig. 7
).
|
It is not obvious how knowledge of ligand selective efficacy would be
applied to drug discovery. However, it could be useful to classify
agonists on the basis of stimulus-response coupling as knowledge for
retrospective analysis. Currently, agonists are all assumed uniformly
to stimulate receptors and differ only on a spectrum of strength of
signal. Separating agonists, in terms of the stimulus-pathways that
they preferentially activate, may offer insights into preferred
profiles of agonism as compounds are progressed from screening assays
into therapeutically oriented secondary assays (104)
.
There are other realms of ligand-selective receptor conformation
selection that may have therapeutic utility. For example, ligands that
selectively induce receptor internalization may have great utility in
the prevention of HIV-1 infection through chemokine receptor fusion.
Ligands that cause internalization of CXCR4 (105
, 106)
or
CCR5 (107
, 108)
have been shown to protect against HIV-1
infection in vitro. The selective removal of chemokine
receptor from the cell surface could be superior to blocking chemokine
receptor interaction with HIV viral coat proteins because it would
circumvent possible rapid emergence of resistant HIV variants through
therapeutic pressure and mutation (109
110
111)
.
There are other realms where differential conformations leading to
differences in receptor disposition could be useful therapeutically.
For example, ligand-selective bias in the production of receptor
desensitization could be beneficial in treatment of tolerance
(99
, 100)
. Similarly, receptor dimerization may be
implicated in numerous areas including HIV-1 infection (112
, 113)
and the function of cannabinoid receptors
(114)
, GABAB receptors
(115
116
117
118)
, adenosine A1 receptors (119)
,
-opioid receptors (120)
,
ß2-adrenoreceptors (121)
, and
calcium-sensing receptors (122
123
124)
. Ligands that induce
selective conformations affecting dimerization may produce unique
effects not necessarily associated with direct receptor signaling.
Finally, it is becoming evident that GPCRs can associate with other
membrane proteins to change their affinities to ligands and
reactivities toward G-proteins. For example, receptor
activity-modifying proteins can change the phenotype of calcitonin
gene-related peptide, adrenomedullin receptors, and calcitonin
receptors (125
126
127
128
129)
. Similarly, GPCRs are known to
interact with other accessory proteins such as PDZ domain-containing
proteins. Thus, ß2-adrenoreceptors interact
with Na+/H+-exchanger
regulatory factor (130)
and 5-HT2C
receptors with MUPP1 (a multi-PDZ domain protein with no currently
known function (131)
. Again, as with desensitization,
dimerization, and internalization, these receptor functions could, in
theory, be regulated differentially by different ligand-induced
receptor conformations to change receptor function. This could lead to
another dimension in control of the quality of ligand efficacy.
|
CONCLUSIONS |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTNEW MOLECULAR TARGETS FOR...INVERSE AGONISTSPROTEAN AGONISTSLIGAND-SPECIFIC RECEPTOR ACTIVE...CONCLUSIONSREFERENCES |
|---|
-opioid receptors coupled to GTP-binding proteins. Proc. Natl. Acad. Sci. USA 86,7321-73252-adrenergic receptor activation of G-proteins: evidence for a pre-coupled receptor/G-protein state. Mol. Pharmacol. 45,524-531[Abstract]
1B-adrenergic receptor by all amino acid substitutions at a single site. J. Biol. Chem. 267,1430-14331b-adrenergic receptor is initiated by disruption of an interhelical salt bridge constraint. J. Biol. Chem. 271,28318-283231a- and 1b-adrenergic receptor subtypes. Mol. Pharmacol. 56,858-8661B- and 1D-adrenoceptors by agonists and inverse agonists. Mol. Pharmacol. 57,659-6661B-adrenoceptor
gene (GNAS1) in a patient with Albright hereditary osteodystrophy. Genomics 13,1319-1321
1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc. Natl. Acad. Sci. 88,11354-11358 2-adrenergic receptor and have a Phe substitute in the G-protein-activator sequence motif. FEBS Lett 311,29-32[Medline]
2A-adrenergic receptors: distinct basic residues mediate Gi versus Gs activation. Mol. Pharmacol. 56,1005-1013 contains mutations in the envelope gp120 but does not show a switch in coreceptor use. J. Virol. 72,4032-4037 opioid receptor. J. Biol. Chem. 272,26959-26964This article has been cited by other articles:
|
|
 |
|
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 |
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|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
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|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
R. R. Neubig Missing Links: Mechanisms of Protean Agonism Mol. Pharmacol., May 1, 2007; 71(5): 1200 - 1202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 G. Rossi, V. Gasperi, R. Paro, D. Barsacchi, S. Cecconi, and M. Maccarrone Follicle-Stimulating Hormone Activates Fatty Acid Amide Hydrolase by Protein Kinase A and Aromatase-Dependent Pathways in Mouse Primary Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1431 - 1439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Verzijl, L. Pardo, M. van Dijk, Y. K. Gruijthuijsen, A. Jongejan, H. Timmerman, J. Nicholas, M. Schwarz, P. M. Murphy, R. Leurs, et al. Helix 8 of the Viral Chemokine Receptor ORF74 Directs Chemokine Binding J. Biol. Chem., November 17, 2006; 281(46): 35327 - 35335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
B. Masri, N. Morin, L. Pedebernade, B. Knibiehler, and Y. Audigier The Apelin Receptor Is Coupled to Gi1 or Gi2 Protein and Is Differentially Desensitized by Apelin Fragments J. Biol. Chem., July 7, 2006; 281(27): 18317 - 18326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kenakin Receptors as Microprocessors: Pharmacological Nuance on Metabotropic Glutamate Receptors 1{alpha} Sci. Signal., July 3, 2006; 2006(342): pe29 - pe29. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Gesty-Palmer, M. Chen, E. Reiter, S. Ahn, C. D. Nelson, S. Wang, A. E. Eckhardt, C. L. Cowan, R. F. Spurney, L. M. Luttrell, et al. Distinct beta-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation J. Biol. Chem., April 21, 2006; 281(16): 10856 - 10864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 H. Schuel and L. J. Burkman A Tale of Two Cells: Endocannabinoid-Signaling Regulates Functions of Neurons and Sperm Biol Reprod, December 1, 2005; 73(6): 1078 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-L. Lu, R. Gallagher, R. Sellar, M. Coetsee, and R. P. Millar Mutations Remote from the Human Gonadotropin-releasing Hormone (GnRH) Receptor-binding Sites Specifically Increase Binding Affinity for GnRH II but Not GnRH I: EVIDENCE FOR LIGAND-SELECTIVE, RECEPTOR-ACTIVE CONFORMATIONS J. Biol. Chem., August 19, 2005; 280(33): 29796 - 29803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. C. Kent, K. S. J. Thompson, and L. H. Naylor Development of a Generic Dual-Reporter Gene Assay for Screening G-Protein-Coupled Receptors J Biomol Screen, August 1, 2005; 10(5): 437 - 446. [Abstract] [PDF]
|
|
|
|
|
|
J. M. Mathiesen, T. Ulven, L. Martini, L. O. Gerlach, A. Heinemann, and E. Kostenis Identification of Indole Derivatives Exclusively Interfering with a G Protein-Independent Signaling Pathway of the Prostaglandin D2 Receptor CRTH2 Mol. Pharmacol., August 1, 2005; 68(2): 393 - 402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zhang, D. Mizrachi, F. Fanelli, and D. L. Segaloff The Formation of a Salt Bridge Between Helices 3 and 6 Is Responsible for the Constitutive Activity and Lack of Hormone Responsiveness of the Naturally Occurring L457R Mutation of the Human Lutropin Receptor J. Biol. Chem., July 15, 2005; 280(28): 26169 - 26176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Krueger, D. G. Witte, L. Ireland-Denny, T. R. Miller, J. L. Baranowski, S. Buckner, I. Milicic, T. A. Esbenshade, and A. A. Hancock G Protein-Dependent Pharmacology of Histamine H3 Receptor Ligands: Evidence for Heterogeneous Active State Receptor Conformations J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 271 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Bartlett, K. W. Young, S. R. Nahorski, and R. A. J. Challiss Single Cell Analysis and Temporal Profiling of Agonist-mediated Inositol 1,4,5-Trisphosphate, Ca2+, Diacylglycerol, and Protein Kinase C Signaling using Fluorescent Biosensors J. Biol. Chem., June 10, 2005; 280(23): 21837 - 21846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J H Li, F Sicard, M A Salam, M Baek, J LePrince, H Vaudry, K Kim, H B Kwon, and J Y Seong Molecular cloning and functional characterization of a type-I neurotensin receptor (NTR) and a novel NTR from the bullfrog brain J. Mol. Endocrinol., June 1, 2005; 34(3): 793 - 807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Lagane, S. Ballet, T. Planchenault, K. Balabanian, E. Le Poul, C. Blanpain, Y. Percherancier, I. Staropoli, G. Vassart, M. Oppermann, et al. Mutation of the DRY Motif Reveals Different Structural Requirements for the CC Chemokine Receptor 5-Mediated Signaling and Receptor Endocytosis Mol. Pharmacol., June 1, 2005; 67(6): 1966 - 1976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-L. Baneres, D. Mesnier, A. Martin, L. Joubert, A. Dumuis, and J. Bockaert Molecular Characterization of a Purified 5-HT4 Receptor: A STRUCTURAL BASIS FOR DRUG EFFICACY J. Biol. Chem., May 27, 2005; 280(21): 20253 - 20260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Assmann Plant G Proteins, Phytohormones, and Plasticity: Three Questions and a Speculation Sci. Signal., December 21, 2004; 2004(264): re20 - re20. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Hellstrand, E A Danielsen, V M Steen, A Ekman, E Eriksson, and C L Nilsson The ser9gly SNP in the dopamine D3 receptor causes a shift from cAMP related to PGE2 related signal transduction mechanisms in transfected CHO cells J. Med. Genet., November 1, 2004; 41(11): 867 - 871. [Full Text] [PDF]
|
|
|
|
|
|
N. H. Moniri, D. Covington-Strachan, and R. G. Booth Ligand-Directed Functional Heterogeneity of Histamine H1 Receptors: Novel Dual-Function Ligands Selectively Activate and Block H1-Mediated Phospholipase C and Adenylyl Cyclase Signaling J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 274 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ogilvie, S. Thelen, B. Moepps, P. Gierschik, A. C. da Silva Campos, M. Baggiolini, and M. Thelen Unusual Chemokine Receptor Antagonism Involving a Mitogen-Activated Protein Kinase Pathway J. Immunol., June 1, 2004; 172(11): 6715 - 6722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Takezako, C. Gogonea, Y. Saad, K. Noda, and S. S. Karnik "Network Leaning" as a Mechanism of Insurmountable Antagonism of the Angiotensin II Type 1 Receptor by Non-peptide Antagonists J. Biol. Chem., April 9, 2004; 279(15): 15248 - 15257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. De Deurwaerdere, S. Navailles, K. A Berg, W. P. Clarke, and U. Spampinato Constitutive Activity of the Serotonin2C Receptor Inhibits In Vivo Dopamine Release in the Rat Striatum and Nucleus Accumbens J. Neurosci., March 31, 2004; 24(13): 3235 - 3241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wang, K. M. Raehal, E. T. Lin, J. J. Lowery, B. L. Kieffer, E. J. Bilsky, and W. Sadee Basal Signaling Activity of {micro} Opioid Receptor in Mouse Brain: Role in Narcotic Dependence J. Pharmacol. Exp. Ther., February 1, 2004; 308(2): 512 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Prather Inverse Agonists: Tools to Reveal Ligand-Specific Conformations of G Protein-Coupled Receptors Sci. Signal., January 13, 2004; 2004(215): pe1 - pe1. [Abstract] [Full Text] [PDF]
|
|
|
|
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G. Swaminath, Y. Xiang, T. W. Lee, J. Steenhuis, C. Parnot, and B. K. Kobilka Sequential Binding of Agonists to the {beta}2 Adrenoceptor: KINETIC EVIDENCE FOR INTERMEDIATE CONFORMATIONAL STATES J. Biol. Chem., January 2, 2004; 279(1): 686 - 691. [Abstract] [Full Text] [PDF]
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 F. Apone, N. Alyeshmerni, K. Wiens, D. Chalmers, M. J. Chrispeels, and G. Colucci The G-Protein-Coupled Receptor GCR1 Regulates DNA Synthesis through Activation of Phosphatidylinositol-Specific Phospholipase C Plant Physiology, October 1, 2003; 133(2): 571 - 579. [Abstract] [Full Text] [PDF]
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M. Azzi, P. G. Charest, S. Angers, G. Rousseau, T. Kohout, M. Bouvier, and G. Pineyro {beta}-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors PNAS, September 30, 2003; 100(20): 11406 - 11411. [Abstract] [Full Text] [PDF]
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H. Wei, S. Ahn, S. K. Shenoy, S. S. Karnik, L. Hunyady, L. M. Luttrell, and R. J. Lefkowitz Independent {beta}-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2 PNAS, September 16, 2003; 100(19): 10782 - 10787. [Abstract] [Full Text] [PDF]
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F. Gbahou, A. Rouleau, S. Morisset, R. Parmentier, S. Crochet, J.-S. Lin, X. Ligneau, J. Tardivel-Lacombe, H. Stark, W. Schunack, et al. Protean agonism at histamine H3 receptors in vitro and in vivo PNAS, September 16, 2003; 100(19): 11086 - 11091. [Abstract] [Full Text] [PDF]
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Y.-S. Bae, J. C. Park, R. He, R. D. Ye, J.-Y. Kwak, P.-G. Suh, and S. Ho Ryu Differential Signaling of Formyl Peptide Receptor-Like 1 by Trp-Lys-Tyr-Met-Val-Met-CONH2 or Lipoxin A4 in Human Neutrophils Mol. Pharmacol., September 1, 2003; 64(3): 721 - 730. [Abstract] [Full Text] [PDF]
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D. Skrzydelski, A.-M. Lhiaubet, A. Lebeau, P. Forgez, M. Yamada, E. Hermans, W. Rostene, and D. Pelaprat Differential Involvement of Intracellular Domains of the Rat NTS1 Neurotensin Receptor in Coupling to G Proteins: A Molecular Basis for Agonist-Directed Trafficking of Receptor Stimulus Mol. Pharmacol., August 1, 2003; 64(2): 421 - 429. [Abstract] [Full Text] [PDF]
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P. J. Pauwels, I. Rauly, and T. Wurch Dissimilar Pharmacological Responses by a New Series of Imidazoline Derivatives at Precoupled and Ligand-Activated {alpha}2A-Adrenoceptor States: Evidence for Effector Pathway-Dependent Differential Antagonism J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1015 - 1023. [Abstract] [Full Text] [PDF]
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T. Wurch, E. A. Boutet-Robinet, C. Palmier, F. C. Colpaert, and P. J. Pauwels Constitutive Coupling of a Chimeric Dopamine D2/alpha 1B Receptor to the Phospholipase C Pathway: Inverse Agonism to Silent Antagonism by Neuroleptic Drugs J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 380 - 390. [Abstract] [Full Text] [PDF]
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A. Bisello, M. Chorev, M. Rosenblatt, L. Monticelli, D. F. Mierke, and S. L. Ferrari Selective Ligand-induced Stabilization of Active and Desensitized Parathyroid Hormone Type 1 Receptor Conformations J. Biol. Chem., October 4, 2002; 277(41): 38524 - 38530. [Abstract] [Full Text] [PDF]
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L. Joubert, S. Claeysen, M. Sebben, A.-S. Bessis, R. D. Clark, R. S. Martin, J. Bockaert, and A. Dumuis A 5-HT4 Receptor Transmembrane Network Implicated in the Activity of Inverse Agonists but Not Agonists J. Biol. Chem., July 5, 2002; 277(28): 25502 - 25511. [Abstract] [Full Text] [PDF]
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 H. Gong, H. Sun, W. J. Koch, T. Rau, T. Eschenhagen, U. Ravens, J. F. Heubach, D. L. Adamson, and S. E. Harding Specific {beta}2AR Blocker ICI 118,551 Actively Decreases Contraction Through a Gi-Coupled Form of the {beta}2AR in Myocytes From Failing Human Heart Circulation, May 28, 2002; 105(21): 2497 - 2503. [Abstract] [Full Text] [PDF]
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N. A. Martin, M. B. Ruckle, S. L. VanHoof, and P. L. Prather Agonist, Antagonist, and Inverse Agonist Characteristics of TIPP (H-Tyr-Tic-Phe-Phe-OH), a Selective delta -Opioid Receptor Ligand J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 661 - 671. [Abstract] [Full Text] [PDF]
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R. C. Gensure, P. H. Carter, B. D. Petroni, H. Juppner, and T. J. Gardella Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis J. Biol. Chem., November 9, 2001; 276(46): 42692 - 42699. [Abstract] [Full Text] [PDF]
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