The myocardium-protective Gly-49 variant of the beta 1-adrenergic receptor exhibits constitutive activity and increased desensitization and down-regulation.

The beta(1)-adrenergic receptor (beta(1)AR) is a major mediator of catecholamine effects in human heart. Patients with heart failure who were hetero- or homozygous for the Gly-49 variant of the beta(1)AR (Gly-49-beta(1)AR) showed improved long-term survival as compared with those with the Ser-49 genotype. Here, the functional consequences of this polymorphism were studied in cells expressing either variant. The Gly-49-beta(1)AR demonstrated characteristic features of constitutively active receptors. In cells expressing the Gly-49-beta(1)AR, both basal and agonist-stimulated adenylyl cyclase activities were higher than in cells expressing the Ser-49 variant (Ser-49-beta(1)AR). The Gly-49-beta(1)AR was more sensitive to the inhibitory effect of the inverse agonist metoprolol and displayed increased affinity for agonists. Isoproterenol potency for adenylyl cyclase activation was higher on membranes expressing the Gly-49-beta(1)AR than on those expressing the Ser-49-beta(1)AR. After incubation with saturating concentrations of catecholamines or sustained stimulation, the Gly-49 variant showed a much higher desensitization, which largely prevailed over constitutive activity in terms of cAMP accumulation. The Gly-49-beta(1)AR also displayed a more profound agonist-promoted down-regulation than the Ser-49 variant. The stronger regulation of the Gly-49-beta(1)AR could explain the beneficial effect of the Gly-49 genotypes on survival, further supporting the concept that beta(1)AR desensitization is protective in heart failure.

␤ 1 AR 1 is the principal subtype of ␤-adrenergic receptors (␤ARs) regulating human heart rate and contractility (1). Stimulation of myocardial ␤ 1 AR by catecholamines results in the activation of the heterotrimeric G s protein, which, in turn, activates the adenylyl cyclase (AC) and promotes the production of cAMP. During chronic heart failure (HF) the persistent compensatory increase of catecholamines causes ␤ 1 AR desensitization and down-regulation. The resulting partial loss of ␤ 1 AR function is believed to be an adaptive mechanism to counteract the cardiotoxicity of chronic adrenergic signaling (2). This hypothesis is supported by the fact that ␤ 1 -adrenergic blockade has proved to be effective in the treatment of chronic HF (3,4).
Two loci of allelic polymorphism were recently identified in the coding region of the human ␤ 1 AR at codons 49 and 389 (5)(6)(7)(8), causing amino acid variability within the extracellular N-terminal and the intracellular C-terminal regions, respectively. The two natural variants of the ␤ 1 AR at position 389, Arg-389-␤ 1 AR and Gly-389-␤ 1 AR, displayed different patterns of coupling to the G s protein and of AC activation in vitro, but no correlation was found between either variant and susceptibility for (7), or severity of (9), myocardial disease. In contrast, the allelic distribution of ␤ 1 AR polymorphism at codon 49 was found to be associated with long term survival of patients with chronic HF (5). Patients with the Ser-49 genotype showed a high mortality rate (46%) after 5 years, whereas patients either homozygous or heterozygous for the Gly-49 variant showed a mortality rate of only 23%.
Because of the major role of ␤ 1 AR in both normal and failing myocardium, the beneficial effect of the Gly-49-␤ 1 AR on survival in HF patients might be due to specific features of this variant in terms of receptor signaling and/or regulation. To address this question, we compared the pharmacological and functional properties of Ser-49-␤ 1 AR and Gly-49-␤ 1 AR after stable transfection in human embryonic kidney 293 cells. Our data show that the Gly-49 and Ser-49 variants differ in terms of coupling to the downstream adenylyl cyclase pathway and agonist-dependent regulation. They also provide a plausible explanation for the myocardial-protective effect of the Gly-49-␤ 1 AR in patients with chronic HF.

EXPERIMENTAL PROCEDURES
Construction and Expression of the Ser-49 and Gly-49 Variants of ␤ 1 AR-The deoxyadenosine at position 145 in the coding sequence for Ser-49 of the human ␤ 1 AR gene was changed for a deoxycytidine by site-directed mutagenesis, using the method of Kunkel (10), to generate a codon for glycine at position 49. The mutation was confirmed by dideoxy sequencing. Both Ser-and Gly-encoding ␤ 1 AR cDNAs were subcloned into the mammalian expression vector pcDNA3. Nearly confluent human embryonic kidney 293 cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum, 100 g/ml streptomycin, 100 units/ml penicillin, and 1 mM glutamine at 37°C and transfected with 8 g of vector coding for either the Ser-49-␤ 1 AR or the Gly-49-␤ 1 AR. Clones resistant to 1 mg/ml G418 were screened for ␤ 1 AR expression by radioligand binding assay using 125 I-CYP (Amersham Biosciences, specific activity, 2200 Ci/mmol) as ligand.
Preparation of Human Myocardial Membranes-Fresh human left ventricular myocardium specimens were obtained from two patients with chronic HF during implantation of left ventricular mechanical support. The patients were enrolled in the national registry for dilated cardiomyopathy, approved by the Ethics Committee at Göteborg Uni-versity, and had given informed consent. Crude membranes were prepared by homogenization in ice-cold buffer (30 mM Tris, pH 7.4, 0.6 mM EDTA, with protease inhibitors as described below), filtered through gauze fiber, and immediately analyzed for AC activity.
Receptor Radioligand Binding-Radioligand binding assays were performed on membranes, prepared as described previously (11), or on whole cells in a final volume of 250 l of 25 mM Tris, pH 7.4, 12 mM MgCl 2 , and 0 -400 pM 125 I-CYP. Nonspecific binding was determined in the presence of 10 M alprenolol. Membranes were incubated for 45 min at 30°C, filtered through Whatman GF/C filters, and washed three times with the same ice-cold buffer. Protein concentrations were determined with the BCA protein assay kit (Pierce). Competition binding experiments were performed with 80 pM 125 I-CYP and increasing concentrations of various agonists and antagonists. Ligand affinities were calculated using Prism 3.0 (GraphPad Software, San Diego, CA).
Measurements of Adenylyl Cyclase Activity in Membranes and Intact Cells-Non-exposed cells or cells exposed to 10 M isoproterenol (ISO) and 10 M ascorbic acid or 10 M ascorbic acid alone for 20 min at 37°C were washed three times with ice-cold phosphate-buffer saline and harvested mechanically using a pipette. After centrifugation at 800 ϫ g, cells were resuspended in ice-cold lysis buffer containing protease inhibitors (5 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin, and 10 mg/liter benzamidine) and membranes were prepared (11). The AC activity was determined in 2-5 g of membranes resuspended in 60 l of 30 mM Tris, pH 7.4, 0.6 mM EDTA, 5 mM MgCl 2 , 0.12 mM ATP, 0.053 mM GTP, 0.1 mM cAMP, 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), 2.8 mM phosphoenolpyruvate, 1 mCi of [␣-32 P]ATP, 0.2 unit of pyruvate kinase, 1 unit of myokinase, and [ 3 H]cAMP (ϳ60,000 cpm). Enzyme activity was determined in the absence or presence of 0.1 nM to 100 M ISO or 100 M metoprolol for 15 min at 37°C. Membranes incubated with 10 M forskolin were used as positive controls. The reaction was stopped with 200 l of ice-cold 0.5 M HCl, followed by 5 min of boiling. Samples were buffered with 0.2 ml of 1.5 M imidazole, pH 7.5, and finally applied to Al 2 O 2 columns. [␣-32 P]cAMP was eluted with 3 ml of 10 mM imidazole, pH 7.5, and counted in a ␤-counter (LKB Wallac). Dose-response curves of AC activity were analyzed using Prism 3.0 (GraphPad Software).
Intracellular cAMP was measured in intact cells exposed to the indicated concentrations of ISO, norepinephrine (NE), or metoprolol, in the presence of IBMX for 10 min at 37°C (dose-response experiments), or in the absence of IBMX, for 0 -8 h at 37°C, with fresh ligands added every other hour (time-course experiments). Ascorbic acid (10 M) was added in the incubation medium to prevent catecholamine oxidation. After incubation, cells were washed three times with ice-cold phosphate-buffered saline and lysed in 300 l of ice-cold 5 mM Tris, pH 7.4, 5 mM EDTA. The suspension was boiled for 3 min and centrifuged. The cAMP contained in the supernatant was measured with a [ 3 H]cAMP assay kit (Amersham Biosciences). Protein concentrations were determined with the BCA protein assay kit (Pierce).
Receptor Internalization and Receptor Down-regulation-Receptor internalization was determined by measuring the number of "sequestered" receptors that became inaccessible to the hydrophilic membraneimpermeable ligand CGP-12177A after agonist stimulation, as previously described (12). The total number of receptors was measured using the membrane-permeable 125 I-CYP radioligand, and the amount of intracellular receptors was determined as the number of 125 I-CYP binding sites that could not be displaced by a 3.5-h incubation with 3 M CGP-12177A at 13°C. Surface receptors were calculated as the difference between total and intracellular receptors. Sequestered receptors correspond to the loss of surface receptors induced by agonist stimulation.
Receptor down-regulation was determined in nearly confluent cells incubated with 1 M ISO and 10 M ascorbic acid, for 0 -20 h at 37°C, in culture medium containing 5 g/ml cycloheximide.
Statistical Analysis-Values are mean Ϯ S.E. or mean Ϯ S.D. as given in the figure legends. Significant differences between means were calculated with Student's t test for either paired or unpaired observations. A value of p Ͻ 0.05 was considered statistically significant.

RESULTS
To test the hypothesis that the natural substitution of a serine by a glycine residue in the N-terminal extracellular domain of the ␤ 1 AR may affect its functional properties, the cDNAs of the Gly-49-␤ 1 AR and of the Ser-49-␤ 1 AR were transfected in human embryonic kidney HEK-293 cells. Stable clones expressing variable amounts (ϳ0.5 to ϳ5 pmol/mg) of either ␤ 1 AR variant were selected.
Ligand Binding Properties of Ser-49-␤ 1 AR and Gly-49-␤ 1 AR-Both Ser-49 and Gly-49 receptor variants bound catecholamine agonists with a rank order of potency that is characteristic of the ␤ 1 AR subtype: isoproterenol Ͼ norepinephrine Ͼ epinephrine (data not shown). High and low affinity states for agonists were resolved for both receptor variants (Table I). The percentage of receptors found in the high affinity state for agonists was comparable in the two variants, although the Gly-49-␤ 1 AR displayed a general trend toward a stronger affinity for agonists. The high affinity constant of the Gly-49-␤ 1 AR for NE was significantly lower than that of the Ser-49-␤ 1 AR (50 Ϯ 16 and 137 Ϯ 26 nM, respectively, p Ͻ 0.05) as was its low affinity constant for ISO (182 Ϯ 16 and 268 Ϯ 17 nM, respectively, p Ͻ 0.01). Competition binding experiments with antagonists on membrane fractions did not show any significant difference between receptor variants (Table I).
Ser-49-␤ 1 AR and Gly-49-␤ 1 AR Coupling to G s /Adenylyl Cyclase-␤ 1 ARs are known to couple exclusively to G s proteins, leading to the activation of AC. To investigate whether ␤ 1 AR polymorphism at codon 49 might affect receptor coupling efficacy to G s , basal and ISO-stimulated AC activities were measured in membranes prepared from cell clones expressing various densities of either ␤ 1 AR variant (Fig. 1). Maximal, membrane-associated, AC activity promoted by forskolin was comparable in the two groups of clones (267 Ϯ 59 and 279 Ϯ 57 pmol/mg/min for Ser-49-␤ 1 AR and Gly-49-␤ 1 AR, respectively, n ϭ 6, data not shown). Dose-response curves of Ser-49 and Gly-49 clones for AC activation were compared either at "low" (Fig. 1A) or "high" (Fig. 1B) densities of receptors. In both cases, basal and maximal ISO-dependent AC activities were significantly higher in membranes expressing the Gly-49-␤ 1 AR. In addition, the Gly-49 variant exhibited a significantly higher potency for ISO than the Ser-49 variant (EC 50 of 11.0 Ϯ 4.1 versus 35.0 Ϯ 5.5 nM, p Ͻ 0.05). To confirm this observation, basal and maximal ISO-dependent AC activities were measured in additional cell clones (Fig. 1, C and D). On average, basal AC activity was 4-fold higher in membranes prepared from Gly-49-␤ 1 AR cells than in membranes from Ser-49-␤ 1 AR cells (n ϭ 11, p Ͻ 0.001) (Fig. 1C). Similarly, maximal ISOstimulated AC activity was, on average, 2-fold higher in Gly-49-␤ 1 AR membranes than in Ser-49-␤ 1 AR membranes (n ϭ 11, p Ͻ 0.01) (Fig. 1D). Inverse Agonism of Metoprolol on Ser-49-␤ 1 AR and Gly-49-␤ 1 AR-From the results above, it appears that the replacement of the Ser-49 residue of the human ␤ 1 AR by a Gly residue is associated with striking changes of receptor function, including constitutive activation of basal cAMP production and amplification of the response to ISO. According to previous studies on constitutively active receptors, ␤ 1 -adrenergic inverse agonists would be expected to cause a stronger inhibition of the basal AC activity in Gly-49-␤ 1 AR membranes than in Ser-49-␤ 1 AR membranes. Metoprolol, a ␤ 1 -selective ␤-blocker widely used for the treatment of chronic HF, is a weak inverse agonist of the ␤ 1 AR, as shown in transfected fibroblasts expressing exogenous constitutively active receptors and in transgenic mice overexpressing the ␤ 1 AR (13,14). Basal AC activity was reduced upon incubation with 100 M metoprolol in both Ser-49 and Gly-49 membranes (from 8.3 Ϯ 1.3 to 5.7 Ϯ 0.8 pmol/mg/min, p Ͻ 0.05, and from 25 Ϯ 4.3 to 14 Ϯ 2.4 pmol/mg/min, p Ͻ 0.01, respectively). However, the inhibition was significantly stronger in Gly-49 membranes (42 Ϯ 1.7% versus 29 Ϯ 5.6%, p Ͻ 0.05) ( Table II). The higher inverse agonism of metoprolol on Gly-49-␤ 1 AR was confirmed in intact cells. A 10-min incubation with metoprolol caused a significant decrease of cAMP accumulation only in cells expressing the Gly-49-␤ 1 AR variant (from 54 to 30 pmol/mg, p Ͻ 0.05).
Desensitization of Ser-49-␤ 1 AR and Gly-49-␤ 1 AR-Functional differences between receptor variants may also arise from the duration of the cellular signal they generate, which, in turn, depends on desensitization mechanisms (15). Desensitization was first studied by comparing the AC activity in membranes from cells, expressing ϳ1 pmol of receptor per milligram of protein, pre-stimulated or not with ISO for 20 min. In both cases, receptor desensitization was characterized by a reduc-tion of maximal AC activity and by a rightward shift of doseresponse curves (Fig. 2). Although a 2-fold increase of EC 50 values was observed for both variants, there was a marked difference in the extent to which the two ␤ 1 AR variants were desensitized. Maximal AC activity was reduced by 57 Ϯ 8% in membranes expressing the Gly-49-␤ 1 AR, whereas the reduction was only 19 Ϯ 3% for Ser-49-␤ 1 AR (p Ͻ 0.01). The study of additional cell clones expressing various receptor densities of either ␤ 1 AR variant showed that the different degrees of desensitization observed in these experiments are indeed attributable to the intrinsic properties of each variant (Fig. 3).
To determine whether the different extent of desensitization between the two ␤ 1 AR variants observed in membrane preparations could affect the concentration of cAMP reached in intact cells, dose-response assays of agonist-promoted cAMP accumulation were carried out on cells from the same clones as in Fig.  3. The intracellular cAMP, accumulated during 10-min incubation with the phosphodiesterase inhibitor IBMX alone, was higher in Gly-49 cells than in Ser-49 cells (56 Ϯ 11 versus 25 Ϯ 5.2 pmol/mg, p Ͻ 0.05), which reflected well the differences of basal AC activity found between the two variants (Fig. 4). The intracellular cAMP concentration reached in Gly-49 and Ser-49 cells, after 10-min stimulation with 1 nM ISO (a concentration 10 -30 times below the EC 50 of AC stimulation, as determined in Fig. 1), was comparable. However, after stimulation with higher concentrations of ISO, cAMP was significantly lower in cells expressing the Gly variant than in Ser-49 cells. Similar results were obtained with the natural ligand NE: intracellular cAMP was significant lower in cells expressing the Gly variant upon stimulation with NE at concentrations of 1 M or above (data not shown).
The myocardium of patients with chronic HF is permanently  Ͻ 0.01). B, dose-response curves of ISO in membranes of higher receptor density, 5.5 pmol/mg Ser-49-␤ 1 AR (E) and 3.6 pmol/mg Gly-49-␤ 1 AR (q). From the dose-response curves, calculated EC 50 values for ISO were 11 Ϯ 5 (n ϭ 5) and 9.1 nM (one experiment in triplicate) in A and B, respectively, for Gly-49-␤ 1 AR and 36 Ϯ 6.4 (n ϭ 7) and 29 nM (one experiment in triplicate) in A and B, respectively, for Ser-49-␤ 1 AR. C, comparison of basal AC activity in cell clones expressing various densities of the Gly-49-␤ 1 AR (q) or the Ser-49-␤ 1 AR (E); on average, basal activity was 4-fold (3.9 Ϯ 0.8) higher in Gly-49 cells than in Ser-49 cells (p Ͻ 0.001). D, comparison of ISO-stimulated maximal AC activity in cells expressing various densities of the Gly-49-␤ 1 AR (q) or the Ser-49-␤ 1 AR (E); on average, maximal stimulated activity was 2-fold (1.9 Ϯ 0.2) higher in Gly-49 cells than in Ser-49 cells (p Ͻ 0.01). Maximal forskolin activity was similar between Ser-49-␤ 1 AR and Gly-49-␤ 1 AR membranes (267 Ϯ 59 and 279 Ϯ 57 pmol/mg/min, n ϭ 5 and n ϭ 6, respectively). Values are mean Ϯ S.E. subjected to increased levels of endogenous catecholamines. We investigated whether, in the context of sustained stimulation with ISO or NE, the different desensitization profile of ␤ 1 AR variants would result in distinct cAMP responses in the HEK-293 model. Cells expressing either receptor variant were incubated for up to 8 h with saturating concentrations of agonists (Fig. 5). Maximal stimulation with ISO or NE promoted a comparable raise of intracellular cAMP at 2 min, for Ser and Gly (431 Ϯ 40 and 335 Ϯ 30 pmol/mg, respectively, in the presence of ISO; 428 Ϯ 32 and 360 Ϯ 27 pmol/mg, respectively, in the presence of NE). However, for longer incubation times with agonist, a significant decrease of intracellular cAMP was observed in Gly-49-␤ 1 AR cells compared with Ser-49-␤ 1 AR cells. To investigate if sustained stimulation with non-saturating concentrations of natural ligand would also promote a different cAMP accumulation in cells expressing either the Gly-49-␤ 1 AR or the Ser-49-␤ 1 AR variant, cells were exposed to 1 M NE (Fig. 6). In cells expressing the Ser-49 variant, stimulated cAMP levels remained stable throughout the experiment (159 Ϯ 13 and 154 Ϯ 6.8 pmol/mg, at times 10 min and 8 h, respectively). In contrast, stimulated cAMP levels de-creased with time in cells expressing the Gly-49-␤ 1 AR, reaching a maximal decrease of 50% at 8 h (from 140 Ϯ 5 to 67 Ϯ 0.1 pmol/mg, from times 10 min to 8 h).
Taken together, desensitization studies indicate that, in the presence of a sustained stimulation by catecholamines, the signal promoted by the "myocardium-protective" Gly-49-␤ 1 AR is turned off much faster and more completely than that promoted by the Ser-49-␤ 1 AR.
Internalization and Down-regulation of Ser-49-␤ 1 AR and Gly-49-␤ 1 AR-Previous studies have shown that ␤ 1 ARs are much more resistant to ligand-promoted endocytosis than ␤ 2 ARs (11,16). We examined whether the different desensitization profile reported above might be reflected at the level of ␤ 1 AR trafficking. Receptor sequestration assays, based on competition between the membrane-impermeable ligand CGP-12177A and the membrane-permeable radioligand 125 I-CYP,  showed that pretreatment with 10 M ISO for 30 min did not significantly affect the number of surface ␤ 1 ARs in cells expressing the Ser-49 variant, whereas ϳ15% of the Gly-49-␤ 1 AR was sequestered under the same conditions (Fig. 7A). Furthermore, important differences between variants were observed for longer times of exposure to the agonist (Fig. 7B). ISO stimulation of cells expressing Gly-49-␤ 1 AR for 15 min to 20 h resulted in a time-dependent reduction of the total number of receptors, reaching 40% after 20 h (p Ͻ 0.01, n ϭ 5). This down-regulation mainly affected surface receptors (Fig. 7B,  inset). In contrast, the Ser-49-␤ 1 AR was not down-regulated under the same conditions (5.0 Ϯ 10%, p ϭ NS).

DISCUSSION
In the present study we have shown that the polymorphism of the 49th codon of the ␤ 1 AR, which was previously found to be associated with the duration of survival among patients with chronic HF, determines important functional differences between receptor variants.
The Gly-49-␤ 1 AR mediated higher basal and agonist-stimulated AC activities compared with the Ser-49 variant and was more sensitive to the inhibitory effect of the inverse agonist metoprolol. In addition, the Gly-49-␤ 1 AR displayed a moderate but significant increase of affinity for agonists, and ISO showed a higher potency for AC activation in membranes prepared from cells expressing the Gly-49-␤ 1 AR than in Ser-49 membranes. Most properties summarized above are characteristic of constitutively active G protein-coupled receptors (GPCRs).
The marked amplification of the response to the agonist and the moderate changes of K d and K act values are less common, but they were reported for some GPCRs with constitutive activity, such as the Asn-111 mutant of the AT 1A receptor (17).
According to the modified ternary complex model of GPCR activation (18), mutations causing constitutive activation are likely to facilitate the isomerization between active and inactive conformations of the receptor. Although most receptoractivating mutations lie in intracellular regions interacting with G proteins or in adjacent transmembrane domains, they can also occur in extracellular regions. For example, several mutations of the extracellular domain of the thyroid-stimulating hormone receptor were reported to cause constitutive activation of the receptor in patients with toxic adenomas (19). Polymorphism within the N-terminal region was also found for the ␤ 2 AR. The Gly-16-␤ 2 AR, for example, showed a 5-fold increase of agonist sensitivity, although this variant did not display higher basal activity as in the case of the Gly-49-␤ 1 AR. The mechanism, by which the Ser-49 3 Gly substitution facilitates ␤ 1 AR activation, will remain speculative before structural studies. However, it is plausible that, because of the very different conformational preferences of glycine and serine residues (20), the Ser 3 Gly substitution may cause a sliding or a change in the orientation of the adjacent transmembrane domain and, consequently, in the overall conformation of the receptor. This hypothesis is consistent with the current model of GPCR stimulation by peptides, which activate the receptors by interacting with amino acid residues located in their extracellular regions (21).
A second striking feature of the Gly-49 variant of the ␤ 1 AR is its high propensity for desensitization. Increased desensitization was evident for high concentrations of catecholamines and/or sustained stimulation of the receptor. Under these conditions, the level of cAMP was indeed much lower in cells expressing the Gly-49 variant than in cells expressing the less active Ser-49-␤ 1 AR. The association between constitutive activity and increased sensitivity to desensitization is probably not fortuitous, because a number of adrenergic receptor mutants generated in vitro were characterized by structural instability (22), increased phosphorylation (23,24), constitutive desensitization (24), or changes in endocytosis (25). The higher level of Gly-49-␤ 1 AR desensitization might be explained by higher affinities of protein kinase A and/or G protein receptor kinases (GRKs) for this variant, as reported for constitutively active ␤ 2 AR mutants (24). Previous studies indicated that phosphorylation by both protein kinase A and GRKs contribute to ␤ 1 AR desensitization (26). The observation, that the desensitization of the Gly-49-␤ 1 AR was more noticeable in the case of sustained agonist activation or in the presence of agonist concentrations close to or above the K d , suggests that GRKs are involved in this phenomenon, because it is well established that these kinases only phosphorylate receptors that are bound by the agonist.
Sustained activation by the agonist failed to induce any significant down-regulation of the Ser-49-␤ 1 AR, whereas a substantial loss of receptor binding sites was observed in cells expressing the Gly-49 variant. GPCR down-regulation may be caused by the sorting of internalized receptors to the lysosomal compartment where they are degraded by resident proteases. The different down-regulation profile of the two ␤ 1 AR variants might thus be explained by their different internalization rate (no significant internalization for the Ser variant versus 15% internalization for the Gly variant). Alternatively, because of its relatively low level of internalization, the Gly-␤ 1 AR might be directly degraded at the plasma membrane by proteases recognizing the activated conformation of the receptor. Such a mechanism, which does not require receptor internalization, was reported for the ␤ 2 AR (27).
The observation that the frequency of Gly-49-␤ 1 AR genotypes are not different between healthy controls and patients with congestive HF or patients with idiopathic dilated cardiomyopathy (5,9), suggests that the functional properties of this variant are not sufficient, by themselves, to promote a myocardial disease. However, the improved survival of patients with chronic HF and of either a Gly-49/Ser-49 or a Gly-49/Gly-49 genotype, might reflect a direct contribution of the Gly-49-␤ 1 AR to the more favorable prognosis.
In patients with HF, the impairment of cardiac output is compensated by a chronic increase of the sympathetic drive, resulting in permanently high concentrations of blood catecholamines. A number of studies, however, indicated that chronic adrenergic signaling in general and its ␤ 1 AR component in particular, are harmful for myocardial tissue. Norepinephrine, the ␤ 1 AR-selective catecholamine, was found to be cardiotoxic and to produce cardiac myocyte injury at concentrations found in failing human heart (28). In transgenic mice, a 5-to 40-fold overexpression of cardiac ␤ 1 AR was reported to induce heart failure within months (29,30). Similarly, a 3-fold overexpression of G␣ s led to myocardial damage characterized by cellular degeneration, hypertrophy, and sudden death (31). In adult rat myocytes, the ␤ 1 AR mediates apoptotic signal through G s stimulation, whereas the ␤ 2 AR is anti-apoptotic via its coupling to G i proteins (32). In the failing heart, ␤-adrenergic signaling is reduced as a consequence of ␤AR desensitization and down-regulation and of increased activity of G i proteins (33). It was proposed that this loss of ␤-adrenergictransducing capacity might represent a protective/adaptive mechanism against catecholamine overload (2). The rationale for ␤-adrenergic receptor blockade, the current established treatment of compensated chronic HF (3,4), is in close accordance with this concept.
Taken together, these data suggest that the protective effect of the Gly-49-␤ 1 AR in HF patients may depend on the higher sensitivity of this receptor variant to catecholamine-induced desensitization. In the context of the chronic adrenergic stimulation occurring in HF, the increased desensitization of the Gly-49 variant is likely to largely override its basal constitutive activity. This hypothesis is consistent with the much lower level of cAMP measured in cells expressing Gly-49-␤ 1 AR, compared with Ser-49-␤ 1 AR cells, upon sustained receptor activation. In addition, the observation that the inverse agonist metoprolol could decrease basal AC activity and cAMP content in cells expressing the Gly-49-␤ 1 AR suggests that inverse agonists might be more beneficial than neutral ␤-blockers in the treatment of patients with HF, which are homo-or heterozygous for the Gly-49 variant. Interestingly, during the time of the study, we could examine two myocardial specimens, one from a patient with Ser-49-␤ 1 AR (homozygous Ser-49 genotype), the other from a patient with Gly-49 (heterozygous Gly-49/Ser-49 genotype). Metoprolol displayed a significant ␤ 1 AR inverse agonism in myocardial membranes from the patient with Gly-49 but not in membranes from the patient with Ser-49-␤ 1 AR (28 Ϯ 5 versus 5 Ϯ 5% reduction of basal AC activity, p Ͻ 0.005). Furthermore, basal and maximal AC activities were higher in membranes from the Gly-49 patient than in Ser-49 membranes (4 Ϯ 0.2 versus 0.6 Ϯ 0.1 and 13 Ϯ 0.6 versus 2 Ϯ 0.2 pmol/mg/min, respectively). The ␤AR densities were 37 and 30 fmol/mg, respectively. Although these data must be considered with caution due to inherent variability in the human population, they are consistent with the results obtained in transfected cells and with a previous clinical study showing that patients with at least one Gly-49-␤ 1 AR allele tended to respond better to ␤-blockers than patients devoid of this allele. The majority of these patients being treated with the inverse agonist metoprolol (5).
In conclusion, we have reported the first example of polymor- AR (open bars) or the Gly-49-␤ 1 AR (filled bars) (n ϭ 3), as described under "Experimental Procedures." Before agonist stimulation, surface receptors corresponded to 87 and 88% of total receptors, for Ser-49 and Gly-49, respectively. B, total receptors were measured by radioligand binding with 125 I-CYP after long term incubation with ISO (1 M) in cells expressing the Ser-49-␤ 1 AR (E) or the Gly-49-␤ 1 AR (q) (n ϭ 5, p Ͻ 0.01 versus Ser-49). Inset, surface receptors after 10-h stimulation with the agonist (two experiments performed in triplicate). Receptor densities ranged between 3 and 5 pmol/mg for both variants. Similar results were obtained with clones expressing ϳ0.5 pmol/mg of either receptor variant (0 Ϯ 7% versus 30 Ϯ 3% reduction of total receptor numbers, for Ser-49-␤ 1 AR and Gly-49-␤ 1 AR, respectively, p Ͻ 0.05). Values are mean Ϯ S.E. phism associated with constitutive adrenergic receptor activation (13,34) and increased susceptibility to desensitization. Although without known consequences in healthy subjects, the functional properties of the variants may affect long term prognosis of patients with chronic HF.