Hierarchy of Polymorphic Variation and Desensitization Permutations Relative to b 1 - and b 2 -adrenergic Receptor Signaling*

agonists act to lower the frequency of spontaneous activation of bA Rs, 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 which 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.


Introduction
Like a number of other G-protein coupled receptors, the β 1 -and β 2 -adrenergic receptor (β 1 AR, β 2 AR) 1 undergo desensitization during continuous exposure to agonist. Such desensitization occurs maximally after several minutes of agonist exposure and is due to decreased interaction with G s , 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-G s coupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the β 1 AR 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 β 1 AR-Arg389 displays an increase in G s coupling compared to β 1 AR-Gly389 (2). So, one can consider that the human β 1 AR 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 β 2 AR, a polymorphism which results in a substitution of Ile for Thr at amino acid 164 in the fourth transmembrane spanning domain results in a decrease in G s coupling (4). Thus, eight such states can be considered for the β 2 ARs as well, again based on genotype and desensitization status. As opposed to their allelic variants, only the β 1 AR-Gly389 and the β 2 AR-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 by guest on March 22, 2020 http://www.jbc.org/ Downloaded from 6 150,000 x g for 1.5 hrs. 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 x g for 1 hr. Radioligand binding with 125 I-CYP was then carried out with each fraction as described above.

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 mean + standard error.

Results
Expression levels of the two β 1 AR 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 β 2 AR cell lines had similar levels of expression (Thr164=783 ± 88, Ile164=1104 ± 111 fmol/mg). Of note, signaling characteristics were compared between the two β 1 AR 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 1). Our initial goal was to assess the degree of agonistpromoted desensitization for the wild-type β 1 AR (Gly389) and the Arg389 polymorphic receptor, and the wild-type β 2 AR (Thr164) and its variant, Ile164. Concerning the β 1 ARs, we knew from previous studies that basal and agonist-stimulated adenylyl cyclase activities (in absolute values) were higher for the Arg389 β 1 AR compared to 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 β 1 AR-  fig 1b). For the β 2 ARs, we also found that the two polymorphic variants differed in the extent of agonist-promoted desensitization (fig 1c, 1d). However, in contrast to what was observed with the β 1 AR, the hyperfunctional β 2 AR-Thr164 actually underwent decreased desensitization. β 2 AR-Thr164 displayed 26±4.0% desensitization vs 37±4.6% found for Ile164- 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 β 1 AR, these data are shown in figure 2a. As is seen, the influence of genetic variation was such that even after desensitization, the maximal Arg389 receptor function (Arg DI ) was equivalent to the maximal non-desensitized Gly389 variant (Gly CI ). The rank order of activities for the various states for the β 1 AR are: Arg CI >Gly CI =Arg DI >Gly DI >Arg CB >Arg DB >Gly CB ≥Gly DB . For the β 2 AR, since the genetically uncoupled Ile164 receptor underwent an even greater degree of desensitization than the wild-type (figure 1c, d), the heterogeneity in adenylyl cyclase activities due to the various permutations was substantial. The eight states are shown in figure 2b. The rank order was thus: 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 G αs , exposed to the phosphodiesterase inhibitor IBMX, and whole cell cAMP production over the ensuing 30 minutes determined in the absence or presence of various concentrations of the inverse agonists CGP-20712 (β 1 AR) or ICI-118551 (β 2 AR). Here, it is the absolute levels of cAMP that are relevant rather than a percent change. Results are shown in figure 3. The basal levels of cAMP

Discussion
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 α 2C AR and the β 3 AR, 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 (GRKs), or second messenger dependent kinases such as PKA or PKC. 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 β 1 -and β 2 AR, 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 β 1 ARs (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 α helix formed between the seventh transmembrane spanning domain and the membrane anchoring palmitoylated cysteine(s) (14). Given the steric properties of Gly within α helices, and the importance of this region for G-protein binding, its not surprising that functional coupling is different between the Arg and Gly β 1 AR variants. The β 2 AR polymorphism (4) consists of a substitution of Ile for Thr in the fourth transmembrane spanning domain, and also displays altered coupling to G s , likely due to changes in the agonist binding pocket which 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 β 1 AR-Arg389 might render it less likely to desensitize; conversely, since its conformation is more favorable for G s coupling, it could be more sensitive to GRK-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 β 1 AR 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 β 1 AR amounts to eight. For the β 1 AR this is graphically displayed in matrix format with the aforementioned states in figure 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 which has been observed (reviewed in (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, G s 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 β 1 AR or β 2 AR variants, as cAMP levels were . It is also assumed that when desensitized the conformation of the receptor is altered by phosphorylation and β-arrestin interaction (16), and these states are thus denoted as R I *2 , R II *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 β 1 AR or β 2 AR, 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., AR I ) 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 β 1 AR and β 2 AR have been shown to be desensitized. Along with receptor downregulation, 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 (17). Recent studies have shown that β 1 AR or β 2 AR polymorphisms are associated with certain physiologic or pathologic phenotypes in human heart failure (18)(19)(20)(21).
However, prior to the current study it has not been clear if 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 β 1 AR and β 2 AR. 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 (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.