Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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The atypical
3-adrenergic receptor (AR) agonist CGP-12177 has
been used to define a novel atypical
-AR subtype, the putative
4-AR. Recent evaluation of recombinant
-AR subtypes
and
-AR-deficient mice, however, has established the identity of the
pharmacological
4-AR as a novel state of the
1-AR protein. The ability of aryloxypropanolamine ligands like CGP-12177 to independently interact with agonist and
antagonist states of the
1-AR has important implications regarding receptor classification and the potential development of
tissue-specific
-AR agonists.
LY-362884; propranolol; catecholamines; phenethanolamines; thermogenesis; lipolysis receptor sites and receptor proteins
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INTRODUCTION |
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Pharmacological data have
accumulated over the past twenty-five years to support the existence of
atypical -adrenergic receptors (AR). A defining feature of these
receptors is the relatively low potency of standard
-AR antagonists,
like propranolol, to suppress receptor activity. Thus catecholamine
stimulation of lipolysis in rat adipocytes occurs in the presence of
concentrations of propranolol that would completely block activation
mediated by the traditional
1- and
2-AR
(3, 9). Nevertheless, the notion of an atypical
2-AR remained controversial until the mid 1980s, when
investigators at then Beecham Pharmaceuticals discovered agonist
compounds that potently activated adipocyte metabolism through a
propranolol-resistant receptor (1, 2). Ultimately, the
"molecular truth" of the adipocyte receptor was revealed upon the
cloning and phamacological analysis of the
3-AR
(4, 6, 7).
Recently, extensive use of 3-AR ligands has revealed the
presence of another atypical receptor, tentatively dubbed the
"
4-AR". In general, three classes of agonists have
been used widely to characterize recombinant
3-AR:
catecholamines, phenethanolamines, and aryloxypropanolamines
(1, 2). Phenethanolamine agonists, like CL-316243, exhibit
high selectivity for
3-AR, and this activity is
resistant to standard beta-blockers. More importantly, CL-316243 fails
to activate adipocyte metabolism in mice lacking
3-AR
(i.e.,
3-AR knockouts), clearly demonstrating the
specificity of the compound for the
3-AR
(31). The prototypical aryloxypropanolamine agonist of
3-AR is CGP-12177, a compound that is central to the pharmacological identification of
4-AR. CGP-12177 was
developed as a
1/
2-AR antagonist and
later was shown to be a partial agonist of native and recombinant
3-AR (7, 8, 22, 30). Like certain other
-AR antagonists, CGP-12177 exhibited weak sympathomimetic activity
at concentrations greater than those required for receptor blockade
(30). The identity of the receptor mediating the
sympathomimetic actions of CGP-12177 was first thought to be the
3-AR, and the observation that CGP-12177-mediated
cardiovascular responses were resistant to standard beta-blockers
supported this hypothesis (11). Nonetheless, the
finding that potent and selective phenethanolamine
3-AR
agonists failed to elicit similar cardiovascular effects suggested that
CGP-12177 interacted with another receptor (12, 14, 20).
Finally, the observation that CGP-12177 stimulates cardiovascular and
thermogenic responses in
3-knockout mice clearly demonstrated that CGP-12177 interacted with a receptor that was not the
3-AR (10, 27).
Before the advent of molecular cloning techniques, receptors were
defined using functional criteria. A central tenet in drug receptor
classification has been that antagonist affinity is independent of
agonist structure (15). Thus the inability of propranolol to potently block CGP-mediated responses in tissues lacking
3-AR was taken as very strong pharmacological evidence
for a novel receptor subtype. When viewed from a strictly functional
perspective, the assertion that antagonist affinity is independent of
agonist structure is almost certainly true. Nevertheless, this
assertion still allows the possibility that a single receptor protein
might adopt multiple active conformations that can be selectively
stabilized by small molecules. In other words, a given receptor protein
can adopt conformations that confer distinct agonist-binding sites.
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THE ![]() ![]() |
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Putative 4-AR exist in tissues, such as fat and
heart, which are known to express high levels of
1-AR
(5, 12-14). Furthermore, one study found that
CGP-12177 activated recombinant
1-AR (26). Nonetheless, the relevance of this observation to the classification of
atypical
-AR was discounted because it seemed that overexpression of
1-AR was required to observe agonist activity. Very
recent studies, however, demonstrated that CGP-12177 activates adenylyl cyclase in CHO cells expressing physiological (43 fmol/mg membrane protein) levels of
1-AR (17, 18). Indeed,
the
1-AR is ~20 times more sensitive to CGP-12177 than
3-AR (6, 17, 18) and exhibits nearly the
same maximal response.
Perhaps more significant to the issue of the identity of the
4-AR was the question of antagonist affinity. Using
selective and nonselective standard
-antagonists, Konkar et al.
(18) found that CGP-12177-mediated activation of
recombinant
1-AR was significantly more resistant to
antagonist blockade than was activation by catecholamine agonists.
These and related results demonstrated that the
1-AR
fulfilled the pharmacological criteria for the
4-AR
(23), namely, activation by CGP-12177, resistance to
beta-blockade, and lack of activation by
3-AR-selective
phenethanolamine agonists.
CGP-12177 is nearly a fully thermogenic agonist in 3-AR
knockout mice (10, 27). Work with recombinant
-AR that
we have cited strongly suggested that
1-AR might mediate
these effects in native tissues. Analysis of brown fat adenylyl cyclase
of
3-AR knockout mice indicated that CGP-12177-mediated
agonism occurs through a receptor that is pharmacologically identical
to recombinant
1-AR (17). Moreover, this
receptor phenotype was absent in
1-AR knockout mice.
Indeed, these studies demonstrated that
3-AR-independent activation of brown fat adenylyl cyclase was mediated exclusively by
1-AR. Similar experiments in heart tissue and cultured
cardiomyocytes also indicate that
1-AR mediates the
agonist effects of aryloxypropanolamines (A. A. Konkar and J. G. Granneman, unpublished observations). These observations are
consistent with a recent study showing that responsiveness of the heart
to CGP-12177 correlated with the activity of the
1-AR
(16).
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IMPLICATIONS FOR DRUG-RECEPTOR CLASSIFICATION |
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Current receptor theory models indicate that receptors can adopt
various conformations, ranging from inactive to fully active, and that
ligands exert effects by stabilizing the receptor protein in a given
state. Thus antagonists stabilize inactive conformations, whereas
agonists stabilize the active state (28, 29). Recent studies, however, suggest that a given receptor can exist in more that
one active conformation (24, 28, 29). Experiments with aryloxpropanolamines support the existence of two active sites or
states of the 1-AR (17, 18). One active
state is recognized by catecholamines and exists in dynamic equilibrium
with an inactive state that is recognized with high affinity by
standard beta-blockers (Fig. 1).
Interestingly, CGP-12177 and LY-362884 also have high affinity for this
inactive state (18). In addition, aryloxypropanolamines can occupy a state of the receptor that is distinct from that stabilized by catecholamines. This agonist state apparently does not
convert readily into the antagonist/agonist states recognized by
standard blockers and catecholamines, as evidenced by agonist-dependent differences in antagonist potency.
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As stated above, receptor theory holds that antagonist affinity is independent of agonist structure for a given receptor. Data with CGP-12177 and LY-362884 are certainly consistent with the classical (functional) concept of receptor and support the conclusions that these ligands interact with a novel receptor. Nonetheless, it is now clear that the two receptor sites can be generated from a single receptor protein.
CGP-12177 has been widely used to define the existence of "atypical
-AR" in various tissues. It is now clear that conclusions drawn
from these experiments should be reexamined. For example, the presence
of
3/
4-AR on human fat cells has been
supported almost exclusively from results obtained with CGP-12177 (for
fuller discussion, see Refs. 17, 18).
Clearly, activation by CGP-12177 (or LY-362884) and resistance of that
activity to blockade by propranolol cannot be used to define atypical
receptor proteins. In this regard, propranolol resistance to
catecholamine activation is a defining feature of
3-AR
activity, and data from experiments using these conditions indicate
that human lipolysis is mediated solely by traditional
1/
2-AR (5, 19, 33).
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UNRESOLVED ISSUES AND IMPLICATIONS FOR DRUG DISCOVERY |
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Many key questions remain. First, the molecular basis for
aryloxypropanolamine agonism is not known. The observation that the
intrinsic activity of CGP-12177 can vary among tissues and cells
expressing similar numbers of -AR suggests that this sensitivity may
involve interactions with other proteins. One possibility is
oligomerization, which has been demonstrated in a growing number of G
protein-coupled receptors and has been shown to alter the pharmacological properties of some of these receptors
(25). It is also conceivable that the aryloxypropanolamine
sensitivity involves interaction of
1-AR with accessory
proteins, perhaps in a manner similar to receptor activity modifying
proteins (21, 24). It is also unknown whether the active
conformations that are stabilized by catecholamines and
aryloxypropanolamines differ in their signaling properties. For
example, differences in G protein-coupling specificity and/or
desensitization properties could greatly influence the quality and
duration of signals generated by aryloxypropanolamine-occupied receptors. Finally, it appears that catecholamines do not activate the
aryloxypropanolamine site. Thus the relevance of this novel activation
state to physiological neurotransmission is entirely uncertain.
Despite these uncertainties, the pharmacology of the atypical
1-AR state is unique enough to have been accepted as a
distinct receptor (i.e., the
4-AR) by an international
sanctioning body. This robust pharmacology raises the question of
whether this receptor state can be exploited for therapeutic benefit.
Of course,
1-AR are present in many tissues, and it
would be desirable to elicit effects that are tissue specific. For
example, potential anti-obesity effects of aryloxypropanolamines
might be achieved through selective activation of adipocyte metabolism.
The ability to generate tissue-specific responses by compounds with
such mixed agonist/antagonist properties will depend on several
factors, including the ambient level of catecholamine stimulation as
well as the proportion of receptors that can assume the two active
conformations. Thus aryloxypropanolamine agonism would be greatest in
tissues expressing high levels of the novel state of the
1-AR and relatively low levels of ambient catecholamine
stimulation. In this regard, we have found that in vitro responsiveness
to CGP-12177 is substantially greater in brown fat than in heart
(A. A. Konkar and J. G. Granneman, unpublished observations).
Indeed, in vivo, CGP-12177 appears to act mainly as an antagonist of
heart rate, but it is also a strong agonist of brown fat thermogenesis.
With knowledge of how the different agonist states are generated, it
may be possible to identify compounds that selectively activate an
aryloxypropanolamine site without affecting catecholamine action. The
selective stabilization of distinct agonist/antagonist states for the
generation of tissue-selective responses is an expanding area of
research in the nuclear receptor field, giving rise to the concept of
selective estrogen receptor modulators. It now seems likely that
similar concepts can be extended to
-AR.
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ACKNOWLEDGEMENTS |
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I thank Drs. A. A. Konkar, A. Chaudhry, C. Burant, and J. Caro for helpful comments.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46339 and DK-37006.
Address for reprint requests and other correspondence: J. Granneman, Dept. of Psychiatry and Behavioral Neuroscience, 2309 Scott Hall, Wayne State Univ. School of Medicine, Detroit, MI 48201 (E-mail: jgranne{at}med.wayne.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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