Hierarchy of Polymorphic Variation and Desensitization Permutations Relative to beta 1- and beta 2-Adrenergic Receptor Signaling*

Deborah A. RathzDagger , Kimberly N. GregoryDagger , Ying FangDagger , Kari M. Brown§, and Stephen B. LiggettDagger §

From the Departments of Dagger  Pharmacology and § Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0564

Received for publication, June 18, 2002, and in revised form, January 3, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta 1-adrenergic receptor (beta 1AR) or a gain-of-function polymorphic variant (beta 1AR-Arg389), and the beta 2-adrenergic receptor (beta 2AR) or a loss-of-function polymorphic receptor (beta 2AR-Ile164). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta 1AR, the enhanced function of the Arg389 receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta 1AR-Arg389 signaling was equivalent to non-desensitized wild-type beta 1AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta 2AR underwent less desensitization compared with beta 2AR-Ile164, thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Like a number of other G-protein-coupled receptors, the beta 1- and beta 2-adrenergic receptors (beta 1AR and beta 2AR,1 respectively) undergo desensitization during continuous exposure to agonist. Such desensitization occurs maximally after several minutes of agonist exposure and is due to decreased interaction with Gs, which is evoked by receptor phosphorylation (1). Thus the signal transduction of these receptors can be characterized as one of two potential conditions or states, defined here as "control" (no recent exposure to agonist) and "desensitized." However, we have recently shown that an alteration in receptor-Gs coupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the beta 1AR gene in the human population (2, 3) results in either Gly or Arg being encoded at amino acid position 389 of the proximal portion of the cytoplasmic tail. In studies using transfected cells with equivalent expression of the two receptors, the beta 1AR-Arg389 displays an increase in Gs coupling compared with beta 1AR-Gly389 (2). So, one can consider that the human beta 1AR can exist in four agonist-stimulated states: Gly389 control and desensitized, and Arg389 control and desensitized. And, since basal (non-agonist) activity is also affected by these genetic and desensitization processes, eight states can be considered. For the beta 2AR, a polymorphism that results in a substitution of Ile for Thr at amino acid 164 in the fourth transmembrane-spanning domain results in a decrease in Gs coupling (4). Thus, eight such states can be considered for the beta 2ARs as well, again based on genotype and desensitization status. As opposed to their allelic variants, only the beta 1AR-Gly389 and the beta 2AR-Thr164 (the receptors often designated as "wild-type") have been studied in regards to desensitization in recombinantly expressing cells (5, 6). Based on the significant impact that both desensitization and polymorphic variation have on coupling, we considered that control signaling with one variant might even be equivalent to the desensitized signaling of the other. Since inverse agonists act to lower the frequency of spontaneous activation of beta ARs, there is the potential for coupling polymorphisms to influence this response as well. Knowing the hierarchy of these states facilitates understanding the molecular basis of receptor response to therapeutic agents and receptor dysfunction that can occur in pathologic states, where both genetic and post-translational modifications occur together. Such studies also provide for a general model that depicts the interaction of genetic and desensitization mechanisms in G-protein-coupled receptor signaling. To investigate this, we expressed these four receptors in Chinese hamster fibroblasts at equivalent levels and studied the relative effects of these genetic modifications and those of short term agonist-promoted desensitization on receptor function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constructs and Transfections-- Site-directed mutagenesis was performed on the wild-type cDNA templates as described previously so as to mimic the human Arg389 beta 1AR and Ile164 beta 2AR variants (2, 4). Wild-type and polymorphic cDNAs were cloned into the mammalian expression vector pBC12B1. CHW-1102 cells were stably transfected by calcium phosphate precipitation. Positive clones were selected based on resistance to 300 µg/ml G418. Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C, 5% CO2, in 100 µg/ml streptomycin, 100 units/ml penicillin, and 80 µg/ml G418. COS-7 cells were transiently transfected and maintained as described (7).

Radioligand Binding-- Confluent layers of CHW cells were washed three times with cold phosphate-buffered saline, lysed in hypotonic buffer (5 mM Tris, 2 mM EDTA, pH 7.4), and mechanically detached with a rubber policeman in a small volume. The particulates were homogenized with a polytron and then centrifuged at 42,000 × g for 10 min. Pellets were then resuspended in 75 mM Tris, 5 mM MgCl2, 2 mM EDTA, pH 7.4. Expression levels were determined in saturation binding assays. Membranes were incubated with 400 pM 125I-cyanopindolol and 100 µM GTP for 2 h at room temperature with nonspecific binding determined in the presence of 1 µM propranolol. Reactions were stopped by dilution and vacuum filtration over Whatmann glass fiber filters. The percentage of the receptor pool that is expressed at the cell surface was determined exactly as described previously (8). Briefly, cells were homogenized as above, centrifuged at 400 × g for 10 min, and the supernatant layered over a 35% sucrose cushion and centrifuged at 150,000 × g for 1.5 h. The 0-35% interface (light vesicular membranes) and the pellet (plasma membranes) were collected, diluted in 5 mM Tris, 2 mM EDTA, pH 7.4, and centrifuged at 200,000 × g for 1 h. Radioligand binding with 125I-cyanopindolol was then carried out with each fraction as described above.

Adenylyl Cyclase Activities, cAMP Measurements, and Desensitization Protocol-- Confluent monolayers of cells were washed twice with Hanks' balanced salt solution and allowed to equilibrate in fresh DMEM for 30 min at 37 °C, 5% CO2. Cells were then incubated with either a 10 µM concentration of the indicated agonist with 100 µM ascorbic acid or with ascorbic acid alone (control) for 20 min, washed five times with cold phosphate-buffered saline, detached, and membranes prepared as above. Membranes were incubated with 30 mM Tris, pH 7.4, 2 mM MgCl2, 0.8 mM EDTA, 120 µM ATP, 60 µM GTP, 100 µM cAMP, 2.8 mM phosphoenolpyruvate, 2.2 µg myokinase, the indicated concentrations of agonist, and 1 µCi of [alpha -32P]ATP for 30 min at 37 °C as described previously (2). The stop buffer contained a [3H]cAMP standard, which accounted for column recovery. [32P]cAMP was separated from [alpha -32P]ATP chromatographically using alumina columns. Untreated (control) cells bearing the two beta 1ARs are designated Arg389C and Gly389C, while those studied after treatment with a 10 µM concentration of the agonist epinephrine (desensitized) are designated Arg389D and Gly389D. Similarly, control beta 2AR are designated Thr164C and Ile164C, while the desensitized state has the subscript "D." An additional qualifier, based on whether the adenylyl cyclase response is in the absence of agonist (basal, B) or in response to isoproterenol (I), provides for eight different permutations. To ascertain the effects of inverse agonists, COS-7 cells were transfected with the indicated receptors and Galpha s. At confluence, cells in 24-well plates were washed and incubated with 100 µM isobutylmethylxanthine with or without varying concentrations of inverse agonists for 45 min. cAMP produced over this time was quantitated by a competitive immunoassay (Amersham Biosciences).

Miscellaneous-- Protein concentrations were determined by the copper bicinchoninic acid method (9). Curve fitting was carried out with PRISM software (GraphPad, San Diego, CA). Dose-response curves were compared by ANOVA with post-hoc t tests when the p value was <0.05. Other results were compared with paired t tests as indicated. Data are presented as means ± S.E.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression levels of the two beta 1AR variants in the membrane preparations utilized for the adenylyl cyclase assays were 206 ± 16 for Gly389 and 170 ± 22 fmol/mg for Arg389. Likewise, the two beta 2AR cell lines had similar levels of expression (Thr164 = 783 ± 88, Ile164 = 1104 ± 111 fmol/mg). Of note, signaling characteristics were compared between the two beta 1AR variants or between the two beta 2AR variants, but not between subtypes. There was no evidence for relevant intracellular accumulation of either polymorphic variant as determined by radioligand binding of light vesicular membrane and plasma membrane fractions (Table I). Our initial goal was to assess the degree of agonist-promoted desensitization for the wild-type beta 1AR (Gly389) and the Arg389 polymorphic receptor, and the wild-type beta 2AR (Thr164) and its variant, Ile164. Concerning the beta 1ARs, we knew from previous studies that basal and agonist-stimulated adenylyl cyclase activities (in absolute values) were higher for the Arg389 beta 1AR compared with the Gly389 receptor. For the current work, cells in culture were exposed to vehicle or agonist for 20 min, washed, membranes prepared, and adenylyl cyclase activities determined. When desensitization is quantitated as the percent decrease of the response relative to that in the absence of agonist pretreatment, the beta 1AR-Gly389 underwent 21 ± 6.7% agonist-promoted desensitization (Fig. 1a, Table I). No change in the EC50 was observed. The hyperfunctional beta 1AR-Arg389 underwent a greater degree of desensitization compared with its allelic variant, amounting to 34 ± 4.1% desensitization (p < 0.01 versus beta 1AR-Gly389, Fig. 1b). For the beta 2ARs, we also found that the two polymorphic variants differed in the extent of agonist-promoted desensitization (Fig. 1, c and d). However, in contrast to what was observed with the beta 1AR, the hyperfunctional beta 2AR-Thr164 actually underwent decreased desensitization. beta 2AR-Thr164 displayed 26 ± 4.0% desensitization versus 37 ± 4.6% found for Ile164-beta 2AR (p < 0.05).


                              
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Table I
Adenylyl cyclase activation under control and desensitized conditions for the polymorphic beta 1- and beta 2-adrenergic receptors
Results are from five to seven independent experiments. There was a significant relationship between genotype and basal and maximal isoproterenol stimulated adenylyl cyclase activities (p < 0.001 by ANOVA). The subscripts C and D denote activities under control (no agonist pretreatment) and desensitized (with agonist pretreatment) conditions, respectively. Iso = maximal isoproterenol stimulated values.


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Fig. 1.   Agonist-promoted desensitization of polymorphic beta 1AR and beta 2AR. Adenylyl cyclase activities were determined in membranes from CHW cells expressing the indicated receptors. Cells were exposed to vehicle (control) or 10 µM epinephrine (desensitized) for 20 min as described under "Materials and Methods." Data are normalized to the control maximal response after subtraction of basal levels. Shown are results from four experiments. For the beta 1AR, the desensitization was greater for the Arg389 compared with the Gly389 variant (34 ± 4.1% versus 21 ± 6.7%, p < 0.01). For the beta 2AR, the Ile164 receptor displayed greater desensitization than the Thr164 allelic counterpart (37 ± 4.0% versus 26 ± 4.0%, p < 0.05).

Although the above data examine the extent of desensitization as a percentage of the control response, the absolute levels of adenylyl cyclase activities (pmol/min/mg) establish a hierarchy of signal transduction based on genotype and desensitization. For the beta 1AR, these data are shown in Fig. 2a. As is seen, the influence of genetic variation was such that even after desensitization, the maximal Arg389 receptor function (ArgDI) was equivalent to the maximal non-desensitized Gly389 variant (GlyCI). The rank order of activities for the various states for the beta 1AR are: ArgCI > GlyCI = ArgDI > GlyDI > ArgCB > ArgDB > GlyCB >=  GlyDB. For the beta 2AR, since the genetically uncoupled Ile164 receptor underwent an even greater degree of desensitization than the wild-type (Fig. 1, c and d), the heterogeneity in adenylyl cyclase activities due to the various permutations was substantial. The eight states are shown in Fig. 2b. The rank order was thus: Thr164CI > Thr164DI > Ile164CI > Ile164DI > Thr164CB > Thr164DB > Ile164CB = Ile164DB.


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Fig. 2.   Effects of desensitization on absolute activities of adenylyl cyclase stimulation by polymorphic beta 1AR and beta 2AR. Shown are data from experiments described in the legend to Fig. 1 plotted as absolute adenylyl cyclase activities (pmol/min/mg). Subscripts indicate control (C) or desensitization (D) conditions. For both beta 1AR and beta 2AR there was a relationship between genotype, desensitization status, and activities (p < 0.005 by ANOVA). Basal and maximal stimulated activities were all different from their allelic or desensitization counterparts except for beta 1AR-GlyC and ArgD maximal stimulations, which were the same.

We also explored whether the polymorphic variations affected the response to inverse agonists. We considered that the conformational effects of these substitutions might constrain the receptor so that it could not be fully "inactivated" (i.e. decreased spontaneous activation) by the binding of inverse agonists. We were unable to obtain consistent results in CHW cells, likely due to the low levels of basal adenylyl cyclase activity and the relatively low expression levels in the stable lines. Thus COS-7 cells were transfected with the various receptors along with Galpha s, exposed to the phosphodiesterase inhibitor isobutylmethylxanthine, and whole cell cAMP production over the ensuing 30 min determined in the absence or presence of various concentrations of the inverse agonists CGP-20712 (beta 1AR) or ICI-118551 (beta 2AR). Here, it is the absolute levels of cAMP that are relevant rather than a percent change. Results are shown in Fig. 3. The basal levels of cAMP were greater for beta 2AR Thr164 compared with beta 2AR Ile164, as might have been predicted from the membrane studies. Interestingly, a similar difference between the two beta 1ARs, which also might be expected, was not observed. This may be because spontaneous (i.e. non-agonist) activation is less apparent with beta 1AR (10), and thus, differences in the two variants may not be as readily discerned. Regarding the responses to inverse agonists for the beta 1ARs, exposure to CGP-20712 resulted in a dose-dependent decrease in cAMP production for both receptors. The response was identical for both, with minimal cAMP levels of 7.1 ± 0.8 and 7.0 ± 0.9 pmol/mg for Arg389 and Gly389 (n = 5). Similarly, for the beta 2ARs the inverse agonist ICI-118551 lowered cAMP to the same levels for both receptors (Thr164 = 7.4 ± 0.1, Ile164 = 6.5 ± 1.0 pmol/mg, n = 4). These results indicate that the responses to inverse agonists are not influenced by these genetic variations.


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Fig. 3.   Efficacy of inverse agonists acting at polymorphic beta 1AR and beta 2AR. Accumulation of whole cell cAMP was determined over a 45-min period after addition of 100 µM isobutylmethylxanthine in the absence or presence of varying concentrations of the inverse agonist CGP-20712 (beta 1AR) or ICI-118551 (beta 2AR). Shown are the maximal responses from four to five experiments. The absolute levels of cAMP accumulation in the presence of inverse agonist were not different between genotypic variants. Expressions were beta 1AR Arg389 = 4.8 ± 0.6, beta 1AR Gly389 = 3.7 ± 0.7, beta 2AR Thr164 = 2.1 ± 0.34, beta 2AR Ile164 2.2 ± 0.39 pmol/mg.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The dynamic nature of signaling by G-protein-coupled receptors has been considered indicative of the ability of these receptors to rapidly adapt to changes in their signaling environment. Processes such as receptor desensitization are critical for maintenance of homeostasis during normal physiological circumstances, may be compensatory in certain pathological states, or may aberrantly alter signaling and contribute to characteristics of disease states (11). Desensitization may also limit the effectiveness of therapeutically administered agonists (tachyphylaxis). Using recombinantly expressed receptors, the nature of rapid agonist-promoted desensitization of many G-protein-coupled receptors has been explored. Some receptors, such as the alpha 2CAR and the beta 3AR, display little or no rapid desensitization (12, 13). Others display a range of desensitization attributed to various mechanisms including phosphorylation by G-protein-coupled receptor kinases, or second messenger-dependent kinases such as cAMP-dependent protein kinase or protein kinase C. Such comparative studies can be helpful in drug design or understanding selected features of disease states. With the recent elucidation of polymorphic variants of receptors such as the beta 1- and beta 2AR, which have significant functional impact on receptor coupling, the potential interaction between desensitization and genetic variation needs to be considered so as to establish how receptor signaling is influenced by both processes.

Here we have carried out studies with polymorphic beta 1ARs (2), which have either Gly or Arg at amino acid 389. This residue is located in the cytoplasmic portion of the receptor, within a predicted alpha  helix formed between the seventh transmembrane-spanning domain and the membrane-anchoring palmitoylated cysteine(s) (14). Given the steric properties of Gly within alpha  helices, and the importance of this region for G-protein binding, it is not surprising that functional coupling is different between the Arg and Gly beta 1AR variants. The beta 2AR polymorphism (4) consists of a substitution of Ile for Thr in the fourth transmembrane-spanning domain, and also displays altered coupling to Gs, likely due to changes in the agonist binding pocket that affects the conformation of the intracellular loops. A priori, it was not clear whether these polymorphisms would enhance or depress agonist promoted desensitization. For example, the robust signaling of beta 1AR-Arg389 might render it less likely to desensitize; conversely, since its conformation is more favorable for Gs coupling, it could be more sensitive to G-protein-coupled receptor kinase-mediated phosphorylation, which is dependent on the receptor being in the active conformation.

We show here that there is a significant impact of these polymorphisms on agonist-promoted desensitization. In the case of beta 1AR function, the desensitized hyperfunctional variant (Arg389) is equivalent to that of the non-desensitized Gly389 receptor. Since the basal level of signaling, which represents spontaneous conversion to R*, is also relevant, the number of permutations, taking into account basal or agonist stimulation, non-desensitized or desensitized, and two polymorphic variants, for the beta 1AR amounts to eight. For the beta 1AR this is graphically displayed in matrix format with the aforementioned states in Fig. 4, which is useful for considering a more generalized scheme of the role of genetic variation in G-protein-coupled receptor signaling (see below). As is shown, there is considerable variation in basal and agonist-stimulated activities upon stratification by genotype and desensitization status. Such a range of relative signaling efficacy, and the complex interactions between desensitization and genotype, may explain the high degree of interindividual variability in physiologic responses to agonists and antagonists that has been observed (reviewed in Ref. 15). An additional level of signaling can also be found when one considers the response to inverse agonists, which bind receptor and tend to stabilize the R state, and thus minimize spontaneous conversion to R*. As such, Gs-coupled receptors display a decrease in basal adenylyl cyclase activity/cAMP production. Depending on the nature of the polymorphism, the response to inverse agonist could also be affected by genetic variation. Interestingly, despite the other effects of these polymorphisms, differences in inverse agonist efficacy were not observed with either the beta 1AR or beta 2AR variants, as cAMP levels were reduced by inverse agonists to the same absolute levels regardless of genotype. (These values are not incorporated into Fig. 4, since they were determined using a different approach and are not directly comparable.)


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Fig. 4.   Matrix representation of relative adenylyl cyclase activities of beta 1AR stratified by genotype, desensitization status, and agonist stimulation. Results are from the computer fit minimum and maximal values from the mean curves from experiments in figure 2, normalized to agonist-stimulated adenylyl cyclase activities of beta 1AR-Arg389 in the non-desensitized (control) state.

For a general model (Fig. 5) of the potential interaction of uncoupling events due to genetic variation and to desensitization, we have made several assumptions to include polymorphisms with various phenotypic effects on receptor signaling. From prior characterization of agonist, receptor, and G-protein interactions of the polymorphic beta AR (2, 4), we have assumed that the conformation of the receptor is altered by the polymorphisms under study, thus the depiction of two active conformations (RI*1, RII*3) based on the two different receptors (RI and RII). It is also assumed that when desensitized the conformation of the receptor is altered by phosphorylation and beta -arrestin interaction (16), and these states are thus denoted as RI*2 and RII*4. The "basal" (i.e. non-agonist bound) signaling of a receptor, due to spontaneous toggling to an active conformation, is also considered here as relevant, as is the effect of desensitization on basal signaling. (Although not affected with the beta 1AR or beta 2AR, the potential for a coupling polymorphism to alter inverse agonist activity is included in the general model.) The abundance of each of the above species at equilibrium is indicated by the arrows and the subscripted brackets. Within this general model 10 relevant levels of signaling, due to the various states or abundance of a given species, are present at steady state. Of note, other minor species of unknown signaling significance, such as agonist-bound receptor that is not activated (i.e. ARI), are not included.


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Fig. 5.   General model of the potential interaction of coupling polymorphisms and desensitization. Shown are two polymorphic receptors (RI, RII), under conditions of agonist (A) or inverse agonist (IA) occupancy. The desensitized state refers to recent prior exposure to agonist in vivo. Different conformations of the receptor are depicted as *1, *2, *3, and *4, which depend on the effects of the polymorphism and desensitization status. The italicized subscripts outside the brackets indicate the relative abundance of a given species. For example, at steady state, it would be expected that k > j.

From the standpoint of beta AR subtypes in the heart, their functional regulation has been linked to a variety of physiological states in diseases such as heart failure. In human heart failure, myocardial beta 1AR and beta 2AR have been shown to be desensitized. Along with receptor down-regulation, this response is thought to be adaptive in that the pathologically altered heart with limited physiologic and metabolic reserves is protected from constant stimulation by high circulating catecholamines. On the other hand, other studies in genetically altered mice have suggested that some aspects of desensitization of beta AR signaling may be maladaptive in experimental heart failure (17). Recent studies have shown that beta 1AR or beta 2AR polymorphisms are associated with certain physiologic or pathologic phenotypes in human heart failure (18-21). However, prior to the current study it has not been clear whether there was any potentiation, or attenuation, of desensitization events by these polymorphisms. The in vitro data presented here indicate that both desensitization and genetic variation together can serve to set the ultimate level of signaling of beta 1AR and beta 2AR. Indeed, the signaling of some receptors, even in the desensitized state, is equivalent to their non-desensitized allelic variants. Stated another way, genetic variation can have an effect of the same magnitude as that of desensitization. Regarding beta AR in heart failure, this may be particularly important in defining patient subsets, tailoring therapeutic regiments, or in the development of new agents (15, 22). As a general paradigm, we present a model by which genetic variation and desensitization of G-protein-coupled receptor signaling can be considered as multiple states. Although the prevalence of functional polymorphisms within the superfamily is not fully defined (23), many G-protein-coupled receptors have been reported to be polymorphic in their coding regions (24, 25), such that the model may be applicable to multiple diverse signaling events by these receptors.

    ACKNOWLEDGEMENTS

We thank Cheryl Theiss for cell culture and Esther Getz for manuscript preparation.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL22619, HL52318, GM61376, and HD07463.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.

To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Albert Sabin Way, Rm. G-062, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: stephen.liggett@uc.edu.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M206054200

    ABBREVIATIONS

The abbreviations used are: beta 1AR and beta 2AR, beta 1- and beta 2-adrenergic receptors, respectively; Gs, stimulatory guanine nucleotide-binding protein; C, control conditions; D, desensitized conditions; B, basal state of adenylyl cyclase activation; I, isoproterenol stimulated state of adenylyl cyclase activation; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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