Beta AR Research Articles

Variation analysis of β3‐adrenergic receptor and melanocortin‐4 receptor genes in childhood obesity

Decreased energy expenditure and increased food intake are principal causes for obesity. In the present study, genotypes of beta(3)-adrenergic receptor (beta(3)AR) and of melanocortin-4 receptor (MC4R), both of which are believed to have a close link to the cause of obesity, were analyzed and compared with phenotypes of childhood obesity. Thirty-five obese children with moderate to severe obesity were enrolled. Direct sequencing of the MC4R coding region and pinpoint-polymerase chain reaction were used to detect genomic variation in the beta(3)AR gene using peripheral blood-derived DNA. Allele frequency of Trp64Arg variation in the beta(3)AR gene in the obese subjects was 0.16, which is comparable with that in the healthy general population in eastern Asia. Comparison of phenotypical characteristics did not show a significant difference between Trp/Trp and Trp/Arg subjects. It was notable that body height SD was significantly higher in the Trp/Trp than the Trp/Arg subjects (0.93 +/- 1.0 SD vs 0.07 +/- 1.3 SD, P= 0.03). Annual weight gains were far beyond a hypothetical fat gain in an Arg64 heterozygote with decreased energy consumption, suggesting increased food intake in childhood obesity. There was, however, no variation in the MC4R gene despite thorough sequencing of the entire coding region. The Trp64Arg variation in the beta(3)AR gene has no relationship to the degree or the incidence of childhood obesity. The majority of childhood obesity can be characterized as tall stature, more rapid weight gain than that expected by decreased energy expenditure. Further investigation is necessary in regard to the increased food intake as a major cause of childhood obesity.

Allele-Specific Binding of Airway Nuclear Extracts to Polymorphic β2-Adrenergic Receptor 5′ Sequence

Like other intronless G protein-coupled receptor genes, the beta(2)-adrenergic receptor (beta(2)AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5'UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to beta(2)AR 5'-flanking sequence, 3'-biotin-labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only approximately 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which beta(2)AR 5'-flanking polymorphisms affect obstructive lung phenotypes.

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.

β2-Adrenergic receptor polymorphisms and treatment-induced regression of left ventricular hypertrophy in hypertension

Although blood pressure is considered the major determinant of left ventricular hypertrophy in hypertension, genetic variability is increasingly being considered among the factors influencing this complication. beta(2)-Adrenergic receptors (beta(2)ARs) are up-regulated in hypertension and largely polymorphic within the human population. Recently, we have shown that the Glu27 beta(2)AR variant is strongly associated with cardiac hypertrophy in hypertension. The objective of this study is to verify whether this polymorphism also affects hypertrophy regression in response to antihypertensive therapy. In a prospective follow-up study we screened 970 hypertensive patients of Caucasian descent for the Gly16Arg, Gln27Glu, and Thr164Ile beta(2)AR polymorphisms and left ventricular echocardiographic hypertrophy and assigned selected patients to enalapril or atenolol to assess left ventricular hypertrophy regression after 2-year follow-up. Results were stratified according to treatment and the Glu27Gln polymorphism of the beta(2)AR. In cells with stable overexpression of the Glu27 or Gln27 variant of beta(2)AR, we also explored the implications of this polymorphism on hypertrophy-related intracellular signal transduction. Among hypertensive patients, the Gly16 allele was found in 63% of patients and the Glu27 allele was found in 40.6%. Both polymorphisms were in linkage disequilibrium, as expected. Four hundred forty-one hypertrophic hypertensive patients completed the 2-year follow-up. At baseline, patients carrying at least 1 allele of the Glu27 variant presented with a larger cardiac size despite similar blood pressure levels (142.9 +/- 22.5 g/m(2) in Glu27 carriers versus 138.2 +/- 18.4 g/m(2) in Gln27 carriers, P < .02). Blood pressure normalization was achieved by both drugs. At follow-up, compared with the Gln27 patients, the Glu27 patients showed a larger reduction in hypertrophy when treated with enalapril (percent change in left ventricular mass, -6.3% +/- 7.7% in Glu27 carriers versus -2.18% +/- 7.9% in Gln27 carriers; P < .05) but not with atenolol therapy (-2.8% +/- 8.9% in Glu27 carriers versus -2.4% +/- 8.8% in Gln27 carriers, P = not significant). In in vitro studies the activation of p38 and extracellular signal-regulated kinase (ERK-) 1/2 (data not shown) and the activity of the atrial natriuretic factor (ANF) promoter after isoproterenol (INN, isoprenaline) stimulation were larger in Glu27 beta(2)AR overexpressing cells than in Gln27 beta(2)AR overexpressing cells (fold difference compared with unstimulated cells, 9.7 +/- 2.9 for Glu27 beta(2)AR versus 4.2 +/- 0.3 for Gln27 beta(2)AR; P < .05). The Glu27 variant of beta(2)AR enhances hypertension-induced left ventricular hypertrophy. In these patients angiotensin-converting enzyme inhibitors are more efficient than beta-blockers in reducing cardiac size.

An improved rapid genotyping method for the study of beta-2 adrenergic receptor gene polymorphisms using single tube allele specific multiplex PCR

Seventeen single nucleotide polymorphisms (SNPs) have been identified so far, within the beta-2 receptor (beta(2) AR) gene. The presence of so many SNPs within the beta(2) AR gene causes a problem, for those studying beta(2) AR pharmacogenetics, in relation to which SNPs to choose. Most of the work has focused on the three common SNPs within the coding block (alleles 16, 27 and 164) and the techniques developed have been for these three functionally important alleles. We report an improved polymerase chain reaction (PCR)-based method for the simultaneous detection of five functionally important beta(2) AR alleles, namely beta 16A/G, beta utr-20C/T, beta 27C/G, beta utr-47C/T and beta 164C/T. Genomic DNA was used as a template for duplex and triplex PCR to detect the polymorphic sites of the five alleles. DNA sequencing analysis confirmed the specificity of this PCR method. This simplified single-tube multiplexed PCR assay provides an easier, faster and more cost-effective method than those available for studying the specified polymorphisms of the beta(2)AR gene.

Enhanced proliferation of astrocytes from β2‐adrenergic receptor knockout mice is influenced by the IGF system

In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (beta(2)AR). beta(2)AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as compared with wild-type cells. beta(2)AR deletion also regulated molecules of the IGF system. Although IGF-1 levels remained unaltered, IGF-2 and type 1 IGF receptor expression was increased in beta(2)AR knockout cells. Furthermore, conditioned medium from knockout astrocytes contained lower levels of IGF binding protein-2 and -4. Our data suggest a deficit of beta(2)AR on astrocytes, as previously reported in multiple sclerosis, may have implications on proliferative status of astrocytes, a feature that might be attributed to regulation of IGF mitogenic actions.

Mechanisms underlying β2‐adrenoceptor‐mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l-arginine uptake, NOS activity (from l-arginine to l-citrulline conversion), membrane potential (using [(3)H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with betaAR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased l-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of l-arginine uptake by l-lysine inhibited NOS activity and, conversely, blockade of NOS using N(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by l-NAME, suggesting that the increase in l-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l-arginine uptake resulting from NO-mediated membrane hyperpolarization.

Salmeterol Stimulation Dissociates β2-Adrenergic Receptor Phosphorylation and Internalization

Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other beta-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting beta-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of beta-agonists. This effect may not be the same with all beta-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce beta(2)AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and beta-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant beta(2)AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-beta-arrestin2 chimera (EGFP-beta-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-beta-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit beta-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.

β2-adrenergic receptor polymorphisms are associated with asthma and COPD in adults

The beta(2)-adrenergic receptor (beta(2)AR) is a transmembrane protein expressed by airway smooth muscle cells. In vitro studies have shown that polymorphisms at amino acid positions 16 and 27 alter receptor function. The aim of this study was to examine the associations between the beta ( 2 ) AR polymorphisms and risks of asthma, chronic obstructive pulmonary disease (COPD) and respiratory symptoms in a sample of adults. Participants were part of a cross-sectional population-based study of risk factors for respiratory disease. A total of 1,090 Caucasian participants completed a detailed respiratory questionnaire, spirometry, methacholine challenge and measurement of gas transfer. Genotyping for beta ( 2 ) AR polymorphisms at positions 16 and 27 was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method. Haplotype frequencies for the two polymorphisms were estimated using the E-M algorithm. We found the Arg16 homozygotes had an increased risk of COPD (OR 5.13; 95% CI 1.40,18.8), asthma (2.44; 1.12,5.31) and symptoms of wheeze (1.84; 1.02,3.35). The Gln27 homozygotes had an increased risk of asthma (2.08; 1.05,4.13) and bronchial hyperreactivity (BHR) (1.92; 1.07,3.46). The Arg16/Gln27 haplotype was associated with asthma (1.63; 1.12,2.38) and COPD (2.91; 1.42,5.94). The Arg16/Gln27 beta(2)AR haplotype is important in COPD, asthma and BHR, and may be associated with more severe respiratory symptoms in middle-aged and older adults.

α2C-Adrenergic Receptors Exhibit Enhanced Surface Expression and Signaling upon Association with β2-Adrenergic Receptors

The alpha(2C)-adrenergic receptor (alpha(2C)AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of alpha(2C)AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of alpha(2C)AR with more than 25 related GPCRs revealed that only coexpression with the beta(2)-adrenergic receptor (beta(2)AR) increased the surface localization of alpha(2C)AR in human embryonic kidney-293 cells. Coimmunoprecipitation of alpha(2C)AR with beta(2)AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that alpha(2C)AR expressed alone was mainly intracellular, whereas alpha(2C)AR coexpressed with beta(2)AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in alpha(2C)AR binding sites upon coexpression with beta(2)AR, with no apparent change in affinity for alpha(2)AR ligands. Functional assays with the alpha(2)AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of beta(2)AR with alpha(2C)AR enhanced alpha(2C)AR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced alpha(2C)AR internalization in response to alpha(2)AR agonists when alpha(2C)AR and beta(2)AR were coexpressed. In addition, substantial cointernalization of alpha(2C)AR in response to betaAR agonists was observed when alpha(2C)AR was coexpressed with beta(2)AR. These data reveal that alpha(2C)AR can interact with beta(2)AR in cells in a manner that regulates alpha(2C)AR surface expression, internalization, and functionality.

Airway smooth muscle prostaglandin-EP1 receptors directly modulate 2-adrenergic receptors within a unique heterodimeric complex

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(2)AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP(1) receptor, since its endogenous agonist prostaglandin E(2) is abundant in the airway, but its functional implications are poorly defined. Activation of EP(1) failed to elicit ASM contraction in mouse trachea via this G(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), an effect accentuated by EP(1) agonist activation. Thus alone, EP(1) does not appear to have a significant direct effect on airway tone but acts as a modulator of the beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma.

Functional β-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes

We sought to determine if different beta-adrenergic receptor (betaAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Employing enriched nuclear preparations, we assayed the specific presence of betaAR by measuring 125I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of beta 1AR and beta 3AR but, surprisingly, not the beta 2AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear betaAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta 3AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another beta 3AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta 1AR-selective agonist, xamoterol, nor a beta 2AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear betaAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

Role of the G Protein-coupled Receptor Kinase Site Serine Cluster in β2-Adrenergic Receptor Internalization, Desensitization, and β-Arrestin Translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.

Influence of β2-Adrenergic Receptor Genotype on Airway Function During Exercise in Healthy Adults

In humans, beta(2)-adrenergic receptors (beta(2)ARs) influence airway tone. There are known functional polymorphisms of the beta(2)AR, such as substitution of glycine for arginine at codon 16. We sought to determine if this variation in genotype differentially influences airway function during exercise. Healthy subjects without asthma who were either homozygous for Arg16 (n = 16; mean age, 29 +/- 2 years [+/- SD]; mean maximum oxygen uptake [Vo(2)], 32 +/- 2 mL/kg/min) or the Gly16 allele (n = 26; mean age, 30 +/- 1 years; mean maximum Vo(2), 33 +/- 1 mL/kg/min) participated in the study. Baseline testing included spirometry and maximal symptom-limited exercise. On a separate day, an arterial cannula was placed to measure catecholamine levels. Subjects then performed exercise at two work levels (40% and 75% of peak work) for 9 min each and performed spirometry at 3-min intervals for assessment of airway function. There were no statistically significant differences between groups in maximum Vo(2) or baseline spirometry (p > 0.05). With both light and heavy exercise, the groups had similar increases in the forced expiratory flow at 50% of vital capacity (FEF(50)). FEF(50) increased by 14 +/- 4% and 15 +/- 3% in arginine and glycine groups, respectively, by end exercise (p > 0.05). During recovery (5 min and 10 min after), the Gly16 homozygotes demonstrated persistent bronchodilation (10 min after FEF(50) = + 7 +/- 2% over pre-exercise) while the Arg16 subjects had a rapid return to baseline (10 min after FEF(50) = - 3 +/- 3%, p = 0.007 between groups). No differences were observed in the catecholamine responses between genotypes, although the increase in epinephrine in the arginine group tended to be higher (p = 0.07). These data suggest that the Arg16Gly polymorphism of the beta(2)AR does not influence airway function during short-duration low- and high-intensity exercise. However, during recovery, the Arg16 genotype is associated with a reduced bronchodilation, possibly due to increased catecholamine desensitization.

THE CATECHOLAMINE–β‐ADRENORECEPTOR–cAMP SYSTEM AND PREDICTION OF CARDIOVASCULAR EVENTS IN HYPERTENSION

Although the importance of elevated circulating plasma catecholamines on cardiac structural and functional remodelling of hypertension is well documented, it is unclear whether the catecholamine-beta-adrenoreceptor (beta AR)-cAMP system can predict different cardiovascular events. 2. A total of 601 identified hypertensive patients with baseline and follow-up plasma levels of noradrenaline (NA) and adrenaline (Adr), lymphocyte beta AR density (B(max)) and intra-lymphocyte cAMP levels in peripheral blood (last examination 60+/-26 months apart) were followed up for an additional 24+/-12 months. 3. After the last follow up, a composite end-point of cardiovascular death, non-fatal myocardial infarction (MI) and stroke occurred in 139 patients (23.1%). In Cox analyses, adjusting for other standard factors as well as treatment effect, NA (hazard ratio 1.22; 95% confidence interval (CI) 1.17-1.28; P=0.0008), Adr (hazard ratio 1.53; 95% CI 1.18-2.00; P=0.002), beta AR (hazard ratio 1.12; 95% CI 1.06-1.17; P=0.007) and cAMP (hazard ratio 1.15; 95% CI 1.09-1.21; P=0.005) separately predicted cardiovascular mortality. Noradrenaline, Adr, beta AR and intra-lymphocyte cAMP separately predicted fatal/non-fatal MI; NA and Adr predicted fatal/non-fatal stroke, whereas B(max) and intra-lymphocyte cAMP levels were not a significant predictor of fatal/non-fatal stroke. When stratifying the study population by NA or Adr (median 4 nmol/L), B(max) (median 600 fmol/10(7) cells) and cAMP (median 5.0 pmol/mg protein) above and below the median values in both parameters categories, patients above the median had composite cardiovascular end-point (all P<0.001) and high cardiovascular death (all P<0.01, log-rank test). 4. These results suggest that plasma NA and Adr are significant predictors of cardiovascular mortality, MI and stroke. The B(max) and intra-lymphocyte cAMP levels are significant predictors of cardiovascular mortality and MI, but not stroke.

Proteomic Analysis of β1-Adrenergic Receptor Interactions with PDZ Scaffold Proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.

The CAREGENE study: polymorphisms of the β1-adrenoceptor gene and aerobic power in coronary artery disease

The heritability of aerobic power and of the response to physical training has been shown in healthy subjects. beta(1)-Adrenergic receptor (beta(1)AR) function affects exercise performance. This study aims to investigate whether the Ser49Gly and Gly389Arg polymorphisms of the beta(1)AR gene or their haplotypes are associated with aerobic power or its response to physical training in coronary artery disease (CAD). Nine hundred and thirty-five biologically unrelated Caucasian patients with CAD who had exercised until exhaustion during graded bicycle testing at baseline and after completion of 3 months of exercise training from 1990 to 2001 (n = 1095) were eligible for inclusion in the CAREGENE (CArdiac REhabilitation and GENetics of Exercise performance) study. Polymorphisms were detected using the invader assay (Third Wave Technologiestrade mark, Madison, Wisconsin, USA). Patients with the Gly49Gly genotype had significantly higher covariate-adjusted aerobic power at baseline than those with Ser49Ser and Ser49Gly (P < 0.05). Adjusted aerobic power at baseline was highest in the Ser49-Gly389/Gly49-Gly389 and Gly49-Arg389/Gly49-Arg389 haplotype combinations. Aerobic power increased significantly (P < 0.001) with physical training. There was no association with the effect of physical training. Ser49Gly and haplotype combinations of Ser49Gly and Gly389Arg of the beta(1)AR gene are associated with aerobic power, but not with the response to physical training in patients with CAD included in the CAREGENE study.

Admixture-matched case-control study: a practical approach for genetic association studies in admixed populations.

Case-control genetic association studies in admixed populations are known to be susceptible to genetic confounding due to population stratification. The transmission/disequilibrium test (TDT) approach can avoid this problem. However, the TDT is expensive and impractical for late-onset diseases. Case-control study designs, in which, cases and controls are matched by admixture, can be an appealing and a suitable alternative for genetic association studies in admixed populations. In this study, we applied this matching strategy when recruiting our African American participants in the Study of African American, Asthma, Genes and Environments. Group admixture in this cohort consists of 83% African ancestry and 17% European ancestry, which was consistent with reports from other studies. By carrying out several complementary analyses, our results show that there is a substructure in the cohort, but that the admixture distributions are almost identical in cases and controls, and also in cases only. We performed association tests for asthma-related traits with ancestry, and only found that FEV(1), a measure for baseline pulmonary function, was associated with ancestry after adjusting for socio-economic and environmental risk factors (P=0.01). We did not observe an excess of type I error rate in our association tests for ancestry informative markers and asthma-related phenotypes when ancestry was not adjusted in the analyses. Furthermore, using the association tests between genetic variants in a known asthma candidate gene, beta(2) adrenergic receptor (beta(2)AR) and DeltaFEF(25-75), an asthma-related phenotype, as an example, we demonstrated population stratification was not a confounder in our genetic association. Our present work demonstrates that admixture-matched case-control strategies can efficiently control population stratification confounding in admixed populations.

Tachyphylaxis to β2-agonists in Spanish asthmatic patients could be modulated by β2-adrenoceptor gene polymorphisms

The study of determinants of asthma is a subject of much interest currently, especially the pharmacogenetic aspects of asthma management. Genetic polymorphisms affecting amino-acids at positions 16 and 27 within beta(2)-adrenoceptor (beta(2)AR) gene have been implicated in the asthma phenotypes and influence on the variability observed in response to use of bronchodilator agents used in the treatment of asthma. Whether these polymorphisms alter the bronchoprotection response to beta(2)-agonist treatment in Spanish asthmatic population is unknown. The aim of this study was to investigate whether genetic polymorphisms within beta(2)AR gene modulate the clinical outcomes of the individual response to beta(2)-agonist therapy and the development of desensitization in Spanish asthmatic patients. In a prospective, case-control study were included 80 asthmatic patients. Based on the standard criteria, patients were classified into two groups: patients with tachyphylaxis and good responders to beta(2)-agonist therapy. DNA samples were genotyped for the Arg(16)Gly and Glu(27)Gln alleles within the beta(2)AR gene as well as in 64 control samples from blood donors. Arg(16) allele was slightly more frequent within the group with tachyphylaxis (P=0.039), whereas Gly(16) allele carriers were overrepresented within the group of good responders (59.7%, P=0.028). On the other hand, the allele frequency of Gln(27) and the proportion of Gln(27) carriers was higher within the group with tachyphylaxis (P=0.010 and 0.049, respectively) and Glu(27) allele carriers were overrepresented within the group of good responders (P=0.026). The Arg(16) and Gln(27) alleles were in strong linkage disequilibrium across this locus, resulting in the occurrence of disease haplotype. The predisposition to develop tachyphylaxis in our population seems to be linked to the Arg(16) and Gln(27) alleles and to the Arg(16)/Gln(27) risk haplotype (positive association between the presence of the Arg(16) and Gln(27) alleles and tachyphylaxis). The Arg(16) allele is perhaps overrepresented due to the strong linkage disequilibrium between both polymorphisms. The presence of the Glu(27) allele seems to be a protective factor against tachyphylaxis in this cohort study.

Activation of Adenylyl Cyclase by Endogenous Gs-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT7Receptor Expression

Human 5-hydroxytryptamine(7) (5-HT(7)) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G(i/o) and G(q/11) are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT(7) receptor expression was sufficient to limit signaling of endogenously expressed G(s)-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT(4(b)) and 5-HT(7(a,b,d)) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G(s)-coupled beta-adrenergic (betaAR) and prostanoid EP (EPR) receptors. betaAR- and EPR-stimulated AC activity was attenuated by 5-HT(7) receptor expression in both membrane preparations and intact HEK293 cells. betaAR- and EPR-stimulated AC activity was unaffected by expression of the G(s)-coupled 5-HT(4) receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) betaAR- and EPR-stimulated AC activity was not restored to control values when Galpha(s) was overexpressed; and 2) beta(1)AR and beta(2)AR activation of Galpha(s) was unaffected by the expression of 5-HT(7) receptors. In addition, overexpression of AC isoforms was unable to rescue betaAR- and EPR-stimulated AC activity. Therefore, 5-HT(7) receptors probably limit access and/or impede activation of AC by betaAR and EP receptors. Although the 5-HT(7) receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.