A Gain-of-function Polymorphism in a G-protein Coupling Domain of the Human beta 1-Adrenergic Receptor*

Deborah A. Mason, J. Donald Moore, Stuart A. Green, and Stephen B. LiggettDagger

From the Departments of Medicine and Pharmacology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The beta 1-adrenergic receptor (beta 1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human beta 1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0.74, respectively), the former previously considered as the human wild-type beta 1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels were markedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5'-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gs and increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5'-(beta ,gamma -imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (RH = 26%). Taken together, these data indicate that this polymorphic variation of the human beta 1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of beta 1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic beta AR agonists and antagonists in cardiovascular and other diseases.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The beta 1-adrenergic receptor (beta 1AR)1 is a member of the adrenergic family of G-protein-coupled receptors, with epinephrine and norepinephrine being endogenous agonists. Like other members of the G-protein-coupled receptor superfamily, the amino terminus is extracellular, the protein is predicted to traverse the cell membrane seven times, and the carboxyl terminus is intracellular. In the adrenergic receptor family, agonists bind in a pocket formed by the transmembrane-spanning domains, and G-protein binding and activation occur at intracellular domains of the loops and tail, typically near the membrane (1). beta 1ARs couple to the stimulatory G-protein, Gs, activating adenylyl cyclase, as well as to non-cAMP pathways such as the activation of ion channels (2). beta 1ARs are expressed on a number of cell types including cardiomyocytes where they serve to increase cardiac inotropy and chronotropy, adipocytes where they mediate lypolysis, and juxtaglomerular cells of the kidney where they regulate renin secretion. It has been known for decades that these responses, as well as those of the beta 2AR, are somewhat variable in the human population (3, 4). Indeed, we have recently found and characterized several beta 2AR polymorphisms that have altered signaling phenotypes compared with wild-type receptor (5-7). This finding has prompted us to examine the beta 1AR coding sequence for variability in the human population. Here we report a common single nucleotide polymorphism resulting in a Gly to Arg switch at intracellular amino acid 389, within a region important for G-protein coupling. The resulting phenotype of the Arg-389 receptor is one of enhanced receptor-Gs interaction, functionally manifested as increased activation of the adenylyl cyclase effector.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Nomenclature-- The wild-type sequence of the intronless human beta 1AR gene is considered that reported by Frielle et al. (8) (GenBankTM accession number J03019). In the current report the adenine of the initiator ATG of the coding block is designated as nucleotide 1, and amino acid 1 is the encoded methionine. The receptor consists of 477 amino acids.

Polymorphism Detection-- Genomic DNA was extracted from blood or banked tissue from normal individuals by standard techniques (9). The study was approved by the University of Cincinnati Institutional Review Board. PCR reactions were established to produce overlapping products spanning 1131 base pairs of the coding region starting from nucleotide 300 of the second transmembrane-spanning domain past the stop codon into the 3'-untranslated region. A consistent deviation from the published sequence was noted at nucleotide 951 where guanine was found in all subjects instead of adenine and likely represents a sequencing error in the original report. Both codons encode for Leu at amino acid 317. At nucleotide 1165, a guanine ("wild-type") or cytosine was found (Fig. 1A), altering the encoded amino acid from Gly to Arg at amino acid position 389. The primers used to amplify the 488-base pair fragment where this polymorphism at position 1165 was detected were: 5'-CAGGAAACAGCTATGACCACTGGAGCCGCCTCTTCGTCTTCTTCAACTG-3' (sense) and 5'-TGGGCTTCGAGTTCACCTGCTATC-3' (antisense). The sense primer contains a 5' M13 forward primer sequence, so that dye primer chemistry using the universal primer could be utilized in the sequencing reactions. PCRs consisted of ~500 ng of DNA, each dNTP at 62.5 nM, 100 pmol of each primer, 0.5 µl of Pwo polymerase (Roche Molecular Biochemicals), 10% of the supplied buffer, and 5% Me2SO in a final volume of 50 µl. Cycling conditions started at 98 °C for 5 min, followed by 98 °C for 45 s, 60 °C for 1 min, and 72 °C for 1 min for 35 cycles, and a final extension of 72 °C for 7 min in a Perkin-Elmer 9600 thermocycler. Automated sequencing was carried out with an Applied Biosystems sequencer using dye primer chemistry. At nucleotide 1165, the G to C transition results in a loss of a BsmF1 restriction endonuclease site. Additional samples were thus studied with the polymorphism identified by the absence or presence of digestion of the aforementioned PCR product with BsmF1 (Fig. 1B).


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Fig. 1.   Genetic variance of the human beta 1AR at nucleotide 1165, amino acid 389. A, PCR fragments generated from genomic DNA were sequenced as described under "Materials and Methods." The G in the indicated position of codon 389 results in a Gly (left-hand sequence), whereas a C encodes for Arg (right-hand sequence). Shown are representative chromatograms from two individuals homozygous for the two alleles. B, PCR fragments were digested with the restriction endonuclease BsmF1, which fails to digest the Arg-389 product because of the G to C transition. Homo., homozygous; control, no enzyme in the reaction.

Constructs and Cell Transfection-- Site-directed mutagenesis was carried out by methods previously described (5) to mimic the Arg-389 polymorphism. Arg-389 and Gly-389 beta 1AR cDNAs were subcloned into the mammalian expression vector pBC12BI (10). CHW-1102 fibroblasts (CHW cells) were permanently transfected with the Gly-389 and Arg-389 beta 1AR constructs by calcium phosphate precipitation as described previously (11). Positive clones were selected based upon resistance to 300 µg/ml G418. Cultures were maintained at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml G418. COS-7 cells were transiently transfected with the beta 1AR constructs and rat Galpha s in pCDNA1 for [35S]GTPgamma S binding studies. These transient transfections were performed by the DEAE-dextran/chloroquine method as described previously (12).

Radioligand Binding-- Confluent monolayers of CHW cells were washed three times with phosphate-buffered saline, lysed in a hypotonic buffer (5 mM Tris, 2 mM EDTA, pH 7.4), detached by scraping with a rubber policeman, and centrifuged at 42,000 × g for 10 min. Crude membranes were resuspended in buffer (75 mM Tris, 12.5 mM MgCl2, 2 mM EDTA, pH 7.4) and saturation binding experiments performed with 125I-CYP as described (12), with nonspecific binding defined by co-incubation with 1 µM propranolol. Reactions for these and other radioligand experiments were terminated by dilution and vacuum filtration over glass fiber filters. One-site agonist competition binding studies were carried out with 40 pM 125I-CYP, 100 µM GTP, and varying concentrations of isoproterenol, epinephrine, or norepinephrine for 2 h at 25 °C. To assess high and low affinity agonist receptor binding, membranes were prepared as described above, except two additional centrifugations were included before the addition of reaction buffer to assure the removal of endogenous GTP. Membranes were incubated with 40 pM 125I-CYP and 18 concentrations of isoproterenol in the presence or absence of the nonhydrolyzable GTP analog Gpp(NH)p at 100 µM for 1 h at 37 °C. Competition data were fit to one-site and two-site models by an iterative least squares technique as described previously (13). A two-site model was considered valid if by F-test the fit was statistically better (p < 0.05) than that obtained with a one-site model.

Adenylyl Cyclase Activity Measurements-- Membranes were incubated with 30 mM Tris, pH 7.4, 2.0 mM MgCl2, 0.8 mM EDTA, 120 µM ATP, 60 µM GTP, 2.8 mM phosphoenolpyruvate, 2.2 µg of myokinase, 100 µM cAMP, and 1 µCi of [alpha -32P]ATP for 30 min at 37 °C as described (11). [32P]cAMP was separated from [alpha -32P]ATP by chromatography over alumina columns. A [3H]cAMP standard included in the stop buffer accounted for individual column recovery. Activities were determined in the presence of vehicle (basal), the indicated concentrations of agonists, or 100 µM forskolin.

[35S]GTPgamma S Binding-- Transiently transfected COS cells were washed three times with phosphate-buffered saline, lysed in hypotonic buffer (5 mM HEPES, 1 mM EDTA, pH 8), detached by scraping with a rubber policeman, and centrifuged for 10 min at 42,000 × g. Pellets were resuspended in the same buffer and centrifuged two additional times. [35S]GTPgamma S binding was carried out by a modification of the method of Befort et al. (14). The reaction consisted of membranes in 10 mM HEPES, pH 7.40, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA buffer, with 1.0 µM GDP and 180 pM [35S]GTPgamma S in the presence of water (basal), 100 µM unlabeled GTPgamma S (to define nonspecific binding), or 10 µM isoproterenol for 2.5 h at 30 °C. Unbound [35S]GTPgamma S was separated using vacuum filtration over glass fiber filters that were washed twice with 10 mM Tris buffer.

Miscellaneous-- Protein concentrations were determined by the copper bicinchoninic acid method (15). Curve fitting was carried out using PRISM software (GraphPad, San Diego, CA). Statistical comparisons were made by paired or unpaired t tests, as appropriate, with significance considered when p < 0.05. Data are presented as mean ± S.E. of the indicated number of independent experiments.

    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Sequencing results from DNA derived from 30 individuals from a defined healthy cohort revealed variance in the examined beta 1AR region (encompassing the coding block from the second transmembrane domain to the last amino acid in the carboxyl terminus of the receptor) in only one location (Fig. 1), at nucleotide 1165, where guanine or cytosine could be found, resulting in a Gly or Arg at amino acid 389. Using BsmF1 restriction digests, additional genotyping at this locus was carried out for a total of 50 samples. While the wild-type human beta 1AR had previously been reported (8) as Gly at amino acid 389, we found a higher frequency of Arg in our normal population. The allele frequencies were 0.74 for Arg and 0.26 for Gly.

To ascertain whether the Arg in this position conferred a distinct phenotype compared with the Gly-389 receptor (which we (11) and others (16) had previously characterized), site-directed mutagenesis was carried out to mimic Arg-389, and the two cDNAs were subcloned into mammalian expression vectors. The final constructs were identical except for the single nucleotide difference. These constructs were used to establish permanent lines of transfected CHW cells, which endogenously lack beta AR expression. As indicated, studies were carried out on several clonal lines from each transfection at matched levels of receptor expression. Radioligand binding studies (Table I) revealed essentially identical dissociation binding constants for 125I-CYP. Competition binding studies with isoproterenol, epinephrine, and norepinephrine in the presence of GTP showed no differences between the two receptors in binding affinities for these agonists.

                              
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Table I
Radioligand binding parameters of the two polymorphic beta 1ARs
Studies were carried out in the presence of 100 µM GTP. ISO, isoproterenol; NE, norepinephrine; EPI, epinephrine.

Basal, isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were determined using membranes from lines expressing Gly-389 at 159 ± 14 fmol/mg and Arg-389 at 151 ± 13 fmol/mg (Fig. 2A). As shown, basal activities were slightly higher with the Arg-389 receptor than with the Gly-389 receptor (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg, n = 4, p < 0.02). The most dramatic differences, however, were seen with agonist stimulation. Maximal isoproterenol-stimulated activities were higher with the Arg-389 receptor than with the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg, n = 4, p < 0.01). Similarly, expressing the responses as -fold increases over basal levels, the Arg-389 receptor displayed a greater stimulation of adenylyl cyclase than the Gly-389 receptor (6.1- ± 0.3-fold versus 3.3- ± 0.1-fold). In contrast, stimulation of adenylyl cyclase by forskolin was not consistently different between the lines (104 ± 11 versus 120 ± 10 pmol/min/mg). Thus, normalizing the data to the extent of stimulation of adenylyl cyclase by forskolin still revealed a significantly greater agonist stimulation by the Arg-389 variant than the Gly-389 variant (Fig. 2B). The EC50 values for isoproterenol stimulation of adenylyl cyclase for Gly-389 and Arg-389 receptors were not different (115 ± 5 versus 132 ± 5 nM). Results from an additional set of studies carried out on other lines at higher expression levels are shown in Fig. 2, C and D (expression was 246 ± 32 and 255 ± 11 fmol/mg for Gly-389 and Arg-389, respectively). Again, the maximal extent of stimulation by agonist was greater with the Arg-389 variant. These differences in adenylyl cyclase stimulation between the two polymorphic receptors were also observed in responses to epinephrine and norepinephrine (data not shown).


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Fig. 2.   Functional coupling of the Gly-389 and Arg-389 receptors to adenylyl cyclase. Shown are the results from studies with clonal lines expressing each receptor at matched levels and the data presented as absolute activities (A) and normalized to the stimulation by forskolin (B). The results of similar studies with two other clonal lines are shown in panels C and D. The Arg-389 demonstrated small increases in basal activities and marked increases in agonist-stimulated activities compared with the Gly-389 receptor. See "Results" for details. Shown are the mean results from four independent experiments carried out with each line. Absent error bars denote that standard errors were smaller than the plotting symbol.

To investigate the basis of these differences between the two receptors, studies were undertaken to assess interactions between agonist, receptor, and Gs with [35S]GTPgamma S binding experiments and agonist competition studies in the absence and presence of Gpp(NH)p. For [35S]GTPgamma S binding, we found that adequate signals were reproducibly obtained with high levels of transient receptor expression (~10 pmol/mg) in COS-7 cells, which were co-transfected with Galpha s. Results of these studies are shown in Fig. 3. Maximal isoproterenol-stimulated [35S]GTPgamma S binding was found to be greater in membranes bearing the Arg-389 versus the Gly-389 receptor, consistent with the adenylyl cyclase studies, which also showed enhanced agonist-stimulated function with the Arg-389 receptor.


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Fig. 3.   [35S]GTPgamma S binding to the two polymorphic beta 1ARs. COS-7 cells were transfected as described under "Materials and Methods." Binding in the presence of 10 µM isoproterenol was greater (p < 0.05) with the Arg-389 than with the Gly-389 receptor. Because of day-to-day variation in the absolute levels of binding, data are presented as a percentage of binding to the wild-type (Gly389) receptor (mean absolute values were 7.7 ± 1.4 × 105 dpm/mg for Gly-389). Basal levels of binding were not different between the two receptors. nt, nontransfected cells.

A well recognized characteristic of agonist binding to receptors such as the beta 2AR is the increase in high affinity binding states, indicative of receptor/G-protein interactions (17-19). Interestingly, we have previously reported (13) that in competition studies carried out in the absence of Gpp(NH)p, agonist-promoted accumulation of a beta 1AR (Gly-389) high affinity fraction is not readily resolved. This result is likely because of the similar affinities of the high and low affinity sites consistent with relatively less free energy transfer during signal transduction. We wondered whether the Arg-389 receptor would in fact have enhanced agonist-promoted high affinity binding, because this receptor has increased functional coupling to Gs. Isoproterenol competition studies were thus carried out in parallel, in the absence and presence of Gpp(NH)p, with washed membranes from CHW cells expressing each receptor. These results are provided in Fig. 4 and Table II. As shown, the displacement curves in the absence of a guanine nucleotide from the Gly-389 experiments were relatively steep (mean pseudo-Hill coefficient, 0.87 ± 0.03) and were best resolved statistically to a one-site model. Gpp(NH)p had no effect on agonist competition with the Gly-389 receptor (Fig. 4). In contrast, the displacement curves obtained with the Arg-389 receptor in the absence of Gpp(NH)p had mean pseudo-Hill coefficients of 0.73 ± 0.03, and the data were best resolved with a two-site model (composite p value < 0.001). The percentage of receptors in the high affinity state with the Arg-389 receptor was 26 ± 1% (Fig. 4 and Table II). As shown, with this receptor the presence of Gpp(NH)p in the reaction resulted in the expected rightward shift, with monophasic, steep curves consistent with a single low affinity agonist interaction.


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Fig. 4.   Agonist competition curves from studies of the Gly-389 and Arg-389 receptors. Experiments were carried out with CHW membranes in the absence and presence of Gpp(NH)p. The Gly-389 data obtained in the absence of a guanine nucleotide was best fit to a one-site model, and the binding was unaffected by Gpp(NH)p. In contrast, studies with Arg-389 resulted in two-site fits with high and low affinity components (see text and Table II) when carried out in the absence of Gpp(NH)p, and curves were right-shifted and represented single-site binding in its presence. Results shown are the mean data from five independent experiments. Absent error bars denote that standard errors were less than the plotting symbol. (Because these experiments were performed at a higher temperature, the Ki values for isoproterenol with Gpp(NH)p are ~4-fold higher than those obtained in the experiments shown in Table I.)

                              
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Table II
Results of agonist (isoproterenol) competition binding studies in the absence and presence of Gpp(NH)p with the Gly-389 and Arg-389 beta 1ARs
The Gly-389 receptor could not be fit to a two-site model with greater significance than a one-site model (see text for details), and thus high and low affinity binding parameters were not obtained. RH, percent of receptors in high affinity states; NA, not applicable.


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These findings reveal in the normal human population the presence of two beta 1AR genetic variants with significant differences in functional signaling. For purposes of consistency, and because of the fact that a number of structure/function studies have been published using the Gly-389 receptor (13, 20), we prefer to continue to designate this receptor as the "wild-type" human beta 1AR, although the Arg variant appears to be more common. This is analogous to the precedent that has been set with the common beta 2AR polymorphisms (5, 6). The site of the variability is ~9 amino acids from the seventh transmembrane-spanning domain, in the intracellular portion of the tail prior to the proposed palmitoylated cysteine(s) (Fig. 5). This region is sometimes referred to as the fourth intracellular loop, but in this report will be termed the proximal portion of the cytoplasmic tail. By analogy with beta 2AR (21, 22), alpha 2AR (23), and other G-protein-coupled receptors, this region is considered important for receptor coupling to its cognate G-protein, Gs. Indeed, the difference between the Arg-389 and Gly-389 receptors is in functional coupling. Receptor-promoted binding of GTPgamma S to Galpha s, another indicator of agonist-initiated coupling to Gs, was similarly different between the two polymorphic beta 1ARs. Consistent with these findings, agonist-promoted accumulation of the high affinity state in washed membrane preparations in the absence of a guanine nucleotide was detected with the Arg-389 receptor but could not be resolved in studies with the Gly-389 receptor. Based on the multistate model (18, 19) of receptor/G-protein interactions, as well as the above results, an elevated basal activity of adenylyl cyclase (i.e. spontaneous toggling to R* in the absence of agonist) should also be expected with the Arg-389 receptor if it has a greater efficiency of stabilizing the active conformation in the presence of agonist, which in fact was the case. The differences in basal activities that we observed were small but consistent, and the magnitude likely results from the relatively low ("physiologic") levels of expression utilized in the functional studies. As such, we consider the functional phenotype as shown in Fig. 2, A and C, to be indicative of signaling in cells that endogenously express the two polymorphic beta 1ARs.


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Fig. 5.   Alignment of amino acid residues of the proximal cytoplasmic tail of the beta 1AR from various species. Note the conservation at the position of the human Gly-Arg polymorphism. Amino acid numbers are from the human receptor.

The residues in this region of the beta 1AR among other species are shown in Fig. 5. The amino acid analogous to position 389 of the human, as well as the surrounding residues, are highly conserved in species sequenced to date, with the only deviation at position 389 being found in the human, where Gly was originally reported. Whereas we know nothing regarding genetic variability in these other species, the high degree of consistency in this region, its importance in G-protein coupling, and the nonconservative (size and charge) nature of the Gly to Arg substitution are consistent with this variation having functional consequences.

As introduced earlier, beta 1AR are expressed on a number of cell types in the body. In the heart, beta 1AR represent the predominant beta AR subtype and is expressed on myocytes of the atria and ventricles, where they act to increase the force and frequency of contraction in response to sympathetic stimulation. It is intriguing to consider the potential role of the beta 1AR polymorphisms at position 389 in regulating cardiac function under pathologic conditions. As left ventricular failure develops, beta 1AR expression and function decrease in human heart failure (24, 25). This response is thought to be a protective mechanism, sparing the heart from sustained sympathetic stimulation in the face of limited metabolic reserves. In earlier phases of the disease, maintenance of beta 1AR function may contribute to improved ventricular function. Given the above circumstances, the dramatic differences in function between the two beta 1AR polymorphisms suggest that the pathophysiology of congestive heart failure may be influenced by the beta 1AR genotype. Indeed, there is unexplained interindividual variation in the progression of heart failure as well as the cardiac response to infused beta -agonists used therapeutically during acute decompensation (26-28). Interestingly, beta AR antagonists (beta -blockers) are utilized in the chronic treatment of heart failure, the presumed basis of which is minimization of the aforementioned consequences of long term sympathetic stimulation. There is, however, significant interindividual variation in the clinical response to beta -blockers in patients with heart failure (29). Based on our current results, it might be predicted that individuals bearing the Arg-389 receptor would be most responsive to beta -blocker therapy because they would have a genetically determined beta 1AR that achieves a greater stimulation of adenylyl cyclase. A similar scenario might be present in the treatment of essential hypertension with beta -blockers, where responders could be predicted by beta 1AR genotyping. These pharmacogenetic concepts will need to be explicitly tested in human studies. While we are unaware of therapeutic agents specifically targeting adipocyte beta 1ARs, the different functional properties of the two beta 1ARs may be the basis of these polymorphisms acting as disease modifiers in obesity. Finally, we have yet to delineate the frequencies of these beta 1AR polymorphisms in various disease groups. Given that both polymorphisms are common in the general population, however, it is unlikely that either represents the primary basis of disease. These polymorphisms may, however, represent small risk factors in common, multifactorial diseases such as heart failure, hypertension, and obesity.

In conclusion, we have found polymorphic variation of the human beta 1AR at amino acid 389, where Gly (previously considered wild-type) or Arg can be found. This variation alters receptor-Gs coupling, manifested as significant differences between the two receptors in agonist-stimulated adenylyl cyclase activation. Such variation may represent a genetic basis for interindividual differences in disease susceptibility or phenotype, or the response to agents targeting the beta 1AR.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL52318 and HL41496.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.

Dagger To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: Stephen.Liggett{at}UC.Edu.

    ABBREVIATIONS

The abbreviations used are: beta AR, beta -adrenergic receptor; Arg-389, beta 1AR polymorphism with Arg at amino acid 389; Gly-389, beta 1AR polymorphism with Gly at amino acid 389; 125I-CYP, 125I-labeled cyanopindolol; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate; GTPgamma S, guanosine 5'-O-(thiotriphosphate); PCR, polymerase chain reaction.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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