INVITED REVIEW
The putative beta 4-adrenergic receptor is a novel state of the beta 1-adrenergic receptor

James G. Granneman

Cellular and Clinical Neurobiology Program, Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan 48201


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The atypical beta 3-adrenergic receptor (AR) agonist CGP-12177 has been used to define a novel atypical beta -AR subtype, the putative beta 4-AR. Recent evaluation of recombinant beta -AR subtypes and beta -AR-deficient mice, however, has established the identity of the pharmacological beta 4-AR as a novel state of the beta 1-AR protein. The ability of aryloxypropanolamine ligands like CGP-12177 to independently interact with agonist and antagonist states of the beta 1-AR has important implications regarding receptor classification and the potential development of tissue-specific beta -AR agonists.

LY-362884; propranolol; catecholamines; phenethanolamines; thermogenesis; lipolysis receptor sites and receptor proteins


    INTRODUCTION
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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 beta -adrenergic receptors (AR). A defining feature of these receptors is the relatively low potency of standard beta -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 beta 1- and beta 2-AR (3, 9). Nevertheless, the notion of an atypical beta 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 beta 3-AR (4, 6, 7).

Recently, extensive use of beta 3-AR ligands has revealed the presence of another atypical receptor, tentatively dubbed the "beta 4-AR". In general, three classes of agonists have been used widely to characterize recombinant beta 3-AR: catecholamines, phenethanolamines, and aryloxypropanolamines (1, 2). Phenethanolamine agonists, like CL-316243, exhibit high selectivity for beta 3-AR, and this activity is resistant to standard beta-blockers. More importantly, CL-316243 fails to activate adipocyte metabolism in mice lacking beta 3-AR (i.e., beta 3-AR knockouts), clearly demonstrating the specificity of the compound for the beta 3-AR (31). The prototypical aryloxypropanolamine agonist of beta 3-AR is CGP-12177, a compound that is central to the pharmacological identification of beta 4-AR. CGP-12177 was developed as a beta 1/beta 2-AR antagonist and later was shown to be a partial agonist of native and recombinant beta 3-AR (7, 8, 22, 30). Like certain other beta -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 beta 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 beta 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 beta 3-knockout mice clearly demonstrated that CGP-12177 interacted with a receptor that was not the beta 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 beta 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.


    THE beta 4-AR IS A NOVEL STATE OF THE beta 1-AR
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Putative beta 4-AR exist in tissues, such as fat and heart, which are known to express high levels of beta 1-AR (5, 12-14). Furthermore, one study found that CGP-12177 activated recombinant beta 1-AR (26). Nonetheless, the relevance of this observation to the classification of atypical beta -AR was discounted because it seemed that overexpression of beta 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 beta 1-AR (17, 18). Indeed, the beta 1-AR is ~20 times more sensitive to CGP-12177 than beta 3-AR (6, 17, 18) and exhibits nearly the same maximal response.

Perhaps more significant to the issue of the identity of the beta 4-AR was the question of antagonist affinity. Using selective and nonselective standard beta -antagonists, Konkar et al. (18) found that CGP-12177-mediated activation of recombinant beta 1-AR was significantly more resistant to antagonist blockade than was activation by catecholamine agonists. These and related results demonstrated that the beta 1-AR fulfilled the pharmacological criteria for the beta 4-AR (23), namely, activation by CGP-12177, resistance to beta-blockade, and lack of activation by beta 3-AR-selective phenethanolamine agonists.

CGP-12177 is nearly a fully thermogenic agonist in beta 3-AR knockout mice (10, 27). Work with recombinant beta -AR that we have cited strongly suggested that beta 1-AR might mediate these effects in native tissues. Analysis of brown fat adenylyl cyclase of beta 3-AR knockout mice indicated that CGP-12177-mediated agonism occurs through a receptor that is pharmacologically identical to recombinant beta 1-AR (17). Moreover, this receptor phenotype was absent in beta 1-AR knockout mice. Indeed, these studies demonstrated that beta 3-AR-independent activation of brown fat adenylyl cyclase was mediated exclusively by beta 1-AR. Similar experiments in heart tissue and cultured cardiomyocytes also indicate that beta 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 beta 1-AR (16).


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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 beta 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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Fig. 1.   Proposed interaction of ligands with the beta 1-adrenergic receptor (AR). beta 1-AR readily converts between active (*) and inactive conformations. Inactive conformations are stabilized with high affinity by typical beta -AR antagonists and by CGP-12177. Conversion to the activated state is induced and/or stabilized by catecholamines. Some portions of receptors also exist in a state whose occupancy by the aryloxypropanolamines (apa) CGP-12177 and LY-362884 leads to receptor activation. How this state arises is unknown, but it does not appear to rapidly equilibrate with states recognized by catecholamines (cat) and typical antagonists.

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 beta -AR" in various tissues. It is now clear that conclusions drawn from these experiments should be reexamined. For example, the presence of beta 3/beta 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 beta 3-AR activity, and data from experiments using these conditions indicate that human lipolysis is mediated solely by traditional beta 1/beta 2-AR (5, 19, 33).


    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 beta -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 beta 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 beta 1-AR state is unique enough to have been accepted as a distinct receptor (i.e., the beta 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, beta 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 beta 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 beta -AR.


    ACKNOWLEDGEMENTS

I thank Drs. A. A. Konkar, A. Chaudhry, C. Burant, and J. Caro for helpful comments.


    FOOTNOTES

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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