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Hierarchy of Polymorphic Variation and Desensitization Permutations Relative to β1- and β2-Adrenergic Receptor Signaling

2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês

10.1074/jbc.m206054200

1083-351X

Deborah A. Rathz, Kimberly N. Gregory, Ying Fang, Kari M. Brown, Stephen B. Liggett,

Protein Kinase Regulation and GTPase Signaling

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 therelative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human β1-adrenergic receptor (β1AR) or a gain-of-function polymorphic variant (β1AR-Arg389), and the β2-adrenergic receptor (β2AR) or a loss-of-function polymorphic receptor (β2AR-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 β1AR, 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 β1AR-Arg389 signaling was equivalent to non-desensitized wild-type β1AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type β2AR underwent less desensitization compared with β2AR-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. 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 therelative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human β1-adrenergic receptor (β1AR) or a gain-of-function polymorphic variant (β1AR-Arg389), and the β2-adrenergic receptor (β2AR) or a loss-of-function polymorphic receptor (β2AR-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 β1AR, 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 β1AR-Arg389 signaling was equivalent to non-desensitized wild-type β1AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type β2AR underwent less desensitization compared with β2AR-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. β1- and β2-adrenergic receptors, respectively stimulatory guanine nucleotide-binding protein control conditions desensitized conditions basal state of adenylyl cyclase activation isoproterenol stimulated state of adenylyl cyclase activation analysis of variance Like a number of other G-protein-coupled receptors, the β1- and β2-adrenergic receptors (β1AR and β2AR,1respectively) 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 (1Liggett S.B. Lefkowitz R.J. Sibley D. Houslay M. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, London1993: 71-97Google Scholar). 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-Gscoupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the β1AR gene in the human population (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 3Moore J.D. Mason D.A. Green S.A. Hsu J. Liggett S.B. Hum. Mutat. 1999; 14: 271Crossref PubMed Scopus (102) Google Scholar) 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 β1AR-Arg389 displays an increase in Gs coupling compared with β1AR-Gly389 (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). So, one can consider that the human β1AR 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 β2AR, 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 (4Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar). Thus, eight such states can be considered for the β2ARs as well, again based on genotype and desensitization status. As opposed to their allelic variants, only the β1AR-Gly389and the β2AR-Thr164 (the receptors often designated as "wild-type") have been studied in regards to desensitization in recombinantly expressing cells (5Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 6Hausdorff W.P. Bouvier M. O'Dowd B.F. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 12657-12665Abstract Full Text PDF PubMed Google Scholar). 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 βARs, 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. Site-directed mutagenesis was performed on the wild-type cDNA templates as described previously so as to mimic the human Arg389 β1AR and Ile164 β2AR variants (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 4Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar). 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 (7McGraw D.W. Forbes S.L. Kramer L.A. Liggett S.B. J. Clin. Invest. 1998; 102: 1927-1932Crossref PubMed Scopus (262) Google Scholar). 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 mmMgCl2, 2 mm EDTA, pH 7.4. Expression levels were determined in saturation binding assays. Membranes were incubated with 400 pm125I-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 (8Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar). 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 with125I-cyanopindolol was then carried out with each fraction as described above. 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 μmconcentration of the indicated agonist with 100 μmascorbic 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 mmTris, pH 7.4, 2 mm MgCl2, 0.8 mmEDTA, 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 [α-32P]ATP for 30 min at 37 °C as described previously (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar). The stop buffer contained a [3H]cAMP standard, which accounted for column recovery. [32P]cAMP was separated from [α-32P]ATP chromatographically using alumina columns. Untreated (control) cells bearing the two β1ARs 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 β2AR 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 Gαs. 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 (AmershamBiosciences). Protein concentrations were determined by the copper bicinchoninic acid method (9Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). 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 thep value was <0.05. Other results were compared with pairedt tests as indicated. Data are presented as means ± S.E. Expression levels of the two β1AR 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 β2AR cell lines had similar levels of expression (Thr164 = 783 ± 88, Ile164 = 1104 ± 111 fmol/mg). Of note, signaling characteristics were compared between the two β1AR variants or between the two β2AR 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 β1AR (Gly389) and the Arg389polymorphic receptor, and the wild-type β2AR (Thr164) and its variant, Ile164. Concerning the β1ARs, we knew from previous studies that basal and agonist-stimulated adenylyl cyclase activities (in absolute values) were higher for the Arg389 β1AR 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 β1AR-Gly389 underwent 21 ± 6.7% agonist-promoted desensitization (Fig.1 a, Table I). No change in the EC50 was observed. The hyperfunctional β1AR-Arg389 underwent a greater degree of desensitization compared with its allelic variant, amounting to 34 ± 4.1% desensitization (p < 0.01 versusβ1AR-Gly389, Fig. 1 b). For the β2ARs, 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 β1AR, the hyperfunctional β2AR-Thr164 actually underwent decreased desensitization. β2AR-Thr164 displayed 26 ± 4.0% desensitization versus 37 ± 4.6% found for Ile164-β2AR (p < 0.05).Table IAdenylyl cyclase activation under control and desensitized conditions for the polymorphic β1- and β2-adrenergic receptorsParametersβ1-Gly389β1-Arg389β2-Thr164β2-IIe164Expression, fmol/mg206 ± 16170 ± 22783 ± 881104 ± 11(% cell surface)(80 ± 2.1)(88 ± 3.5)(85 ± 1.7)(83 ± 2.1)Adenylyl cyclase, pmol/min/mgBasalC3.7 ± 0.618.2 ± 1.14.4 ± 0.892.0 ± 0.25BasalD2.5 ± 0.435.5 ± 0.743.8 ± 0.711.9 ± 0.54IsoC15.1 ± 2.221.8 ± 2.817.1 ± 0.458.9 ± 0.45IsoD11.9 ± 2.314.9 ± 2.213.1 ± 0.566.4 ± 1.29pK actpK actC2.28 ± 0.172.34 ± 0.191.38 ± 0.221.48 ± 0.09pK actD2.32 ± 0.232.50 ± 0.191.08 ± 0.141.28 ± 0.20 Open table in a new tab 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 β1AR, these data are shown in Fig.2 a. 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 β1AR are: ArgCI > GlyCI = ArgDI > GlyDI > ArgCB > ArgDB > GlyCB ≥ GlyDB. For the β2AR, since the genetically uncoupled Ile164 receptor underwent an even greater degree of desensitization than the wild-type (Fig. 1, c andd), the heterogeneity in adenylyl cyclase activities due to the various permutations was substantial. The eight states are shown in Fig. 2 b. The rank order was thus: Thr164CI > Thr164DI > Ile164CI > Ile164DI > Thr164CB > Thr164DB > Ile164CB = Ile164DB. 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 Gαs, 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 (β1AR) or ICI-118551 (β2AR). 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 β2AR Thr164 compared with β2AR Ile164, as might have been predicted from the membrane studies. Interestingly, a similar difference between the two β1ARs, which also might be expected, was not observed. This may be because spontaneous (i.e. non-agonist) activation is less apparent with β1AR (10Engelhardt S. Grimmer Y. Fan G.H. Lohse M.J. Mol. Pharmacol. 2001; 60: 629-631PubMed Google Scholar), and thus, differences in the two variants may not be as readily discerned. Regarding the responses to inverse agonists for the β1ARs, 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 β2ARs 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. 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 (11Bunemann M. Lee K.B. Pals-Rylaarsdam R. Roseberry A.G. Hosey M.M. Annu. Rev. Physiol. 1999; 61: 169-192Crossref PubMed Scopus (134) Google Scholar). 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 α2CAR and the β3AR, display little or no rapid desensitization (12Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 13Liggett S.B. Freedman N.J. Schwinn D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3665-3669Crossref PubMed Scopus (208) Google Scholar). 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 β1- and β2AR, 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 β1ARs (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar), which have either Gly or Arg at amino acid 389. This residue is located in the cytoplasmic portion of the receptor, within a predicted α helix formed between the seventh transmembrane-spanning domain and the membrane-anchoring palmitoylated cysteine(s) (14Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). Given the steric properties of Gly within α 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 β1AR variants. The β2AR polymorphism (4Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar) 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 β1AR-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 β1AR function, the desensitized hyperfunctional variant (Arg389) is equivalent to that of the non-desensitized Gly389receptor. 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 β1AR amounts to eight. For the β1AR 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. 15Liggett S.B. Nat. Med. 2001; 7: 281-283Crossref PubMed Scopus (74) Google Scholar). 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 β1AR or β2AR 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.) 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 βAR (2Mason D.A. Moore J.D. Green S.A. Liggett S.B. J. Biol. Chem. 1999; 274: 12670-12674Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, 4Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar), 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 β-arrestin interaction (16Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar), 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 β1AR or β2AR, 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. From the standpoint of βAR 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 β1AR and β2AR 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 βAR signaling may be maladaptive in experimental heart failure (17Liggett S.B. J. Clin. Invest. 2001; 107: 947-948Crossref PubMed Scopus (41) Google Scholar). Recent studies have shown that β1AR or β2AR polymorphisms are associated with certain physiologic or pathologic phenotypes in human heart failure (18Liggett S.B. Wagoner L.E. Craft L.L. Hornung R.W. Hoit B.D. McIntosh T.C. Walsh R.A. J. Clin. Invest. 1998; 102: 1534-1539Crossref PubMed Scopus (310) Google Scholar, 19Wagoner L.E. Craft L.L. Singh B. Suresh D.P. Zengel P.W. McGuire N. Abraham W.T. Chenier T.C. Dorn II., G.W. Liggett S.B. Circ. Res. 2000; 86: 834-840Crossref PubMed Scopus (156) Google Scholar, 20Wagoner L.E. Craft L.L. Zengel P. McGuire N. Rathz D.A. Dorn G.W.I. Liggett S.B. Am. Heart J. 2002; 144: 840-846Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 21Small K.M. Wagoner L.E. Levin A.M. Kardia S.L.R. Liggett S.B. N. Engl. J. Med. 2002; 347: 1135-1142Crossref PubMed Scopus (493) Google Scholar). 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 vitrodata presented here indicate that both desensitization and genetic variation together can serve to set the ultimate level of signaling of β1AR and β2AR. 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 βAR in heart failure, this may be particularly important in defining patient subsets, tailoring therapeutic regiments, or in the development of new agents (15Liggett S.B. Nat. Med. 2001; 7: 281-283Crossref PubMed Scopus (74) Google Scholar, 22Liggett S.B. Pharmacology. 2000; 61: 167-173Crossref PubMed Scopus (133) Google Scholar). 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 (23Small K.M. Seman C.A. Castator A. Brown K.M. Liggett S.B. FEBS Lett. 2002; 516: 253-256Crossref PubMed Scopus (17) Google Scholar), many G-protein-coupled receptors have been reported to be polymorphic in their coding regions (24Rana B.K. Shiina T. Insel P.A. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 593-624Crossref PubMed Scopus (91) Google Scholar, 25Small K.M. Tanguay D.A. Nandabalan K. Zhan P. Stephens J.C. Liggett S.B. Am. J. Pharmacogenomics. 2003; 3: 65-71Crossref PubMed Scopus (24) Google Scholar), such that the model may be applicable to multiple diverse signaling events by these receptors. We thank Cheryl Theiss for cell culture and Esther Getz for manuscript preparation.