Beta 1/beta 2-adrenergic receptor heterodimerization regulates beta 2-adrenergic receptor internalization and ERK signaling efficacy

Beta(1)- and beta(2)-adrenergic receptors (beta(1)AR and beta(2)AR) are co-expressed in numerous tissues where they play a central role in the responses of various organs to sympathetic stimulation. Although the two receptor subtypes share some signaling pathways, each has been shown to have specific signaling and regulatory properties. Given the recent recognition that many G protein-coupled receptors can form homo- and heterodimers, the present study was undertaken to determine whether the beta(1)AR and beta(2)AR can form dimers in cells and, if so, to investigate the potential functional consequences of such heterodimerization. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we show that beta(1)AR and beta(2)AR can form heterodimers in HEK 293 cells co-expressing the two receptors. Functionally, beta-adrenergic stimulated adenylyl cyclase activity was found to be identical in cells expressing beta(1)AR, beta(2)AR, or both receptors at similar levels, indicating that heterodimerization did not affect this signaling pathway. When considering ERK1/2 MAPK activity, a significant agonist-promoted activation was detected in beta(2)AR- but not beta(1)AR-expressing cells. Similarly to what was observed in cells expressing the beta(1)AR alone, no beta-adrenergic stimulated ERK1/2 phosphorylation was observed in cells co-expressing the two receptors. A similar inhibition of agonist-promoted internalization of the beta(2)AR was observed upon co-expression of the beta(1)AR, which by itself internalized to a lesser extent. Taken together, our data suggest that heterodimerization between beta(1)AR and beta(2)AR inhibits the agonist-promoted internalization of the beta(2)AR and its ability to activate the ERK1/2 MAPK signaling pathway.     

Beta3-adrenergic receptor

The beta3-adrenergic receptor (beta3-AR) may play a key role in the regulation of lipid metabolism and glucose homeostasis. Adrenaline and noradrenaline beta3-AR stimulate lipolysis and thermogenesis in human fat cells and increase glucose uptake in skeletal muscle. Therefore, the beta3-AR gene may be associated with obesity and related diseases, such as type 2 diabetes, coronary heart disease and hypertension. Many studies in different ethnic groups showed an association of beta3-AR gene polymorphism with insulin resistance, obesity and its metabolic disorders such as type 2 diabetes, coronary heart disease and hypertension. A Trp64Arg mutation in the beta3-AR gene has been reported to be correlated with the occurrence of those disorders among obese. Several studies revealed also the influence of the Trp/Arg polymorphism on carcinogenesis and its contribution to the link between cancer and obesity. Since obesity is a serious problem as a civilization-related disease, it is very important to investigate genes suspected to be connected with it.     

Cardiovascular hemodynamics with increasing exercise intensities in postmenopausal women.

We sought to determine the cardiovascular responses to increasing exercise intensities in postmenopausal women with different physical activity levels and hormone replacement therapy (HRT) status. Forty-four women (11 sedentary, 19 physically active, 14 master athletes; 24 not on HRT, 20 on HRT) completed treadmill exercise at 40, 60, 80, and 100% of maximal oxygen consumption. Oxygen consumption, heart rate, blood pressure, and cardiac output, determined via acetylene rebreathing, were measured at each exercise intensity. HRT did not affect cardiovascular hemodynamics. Stroke volume (SV) decreased significantly between 40 and 100% of maximal oxygen consumption in all groups, and the decrease did not differ among groups. The greater oxygen consumption of the athletes at each intensity was due to their significantly greater cardiac output, which was the result of a significantly greater SV, compared with both of the less active groups. The athletes had significantly lower total peripheral resistance at each exercise intensity than did the two less active groups. There were no consistent significant hemodynamic differences between the physically active and sedentary women. These results indicate that SV decreases in postmenopausal women as exercise intensity increases to maximum, regardless of their habitual physical activity levels or HRT status.     

Relationship of beta(3)-adrenergic receptor polymorphism with metabolic syndrome and oxidative stress parameters in postmenopausal women

INTRODUCTION: Some studies indicate, that the Trp(64)/Arg(64) polymorphism of beta(3)-adrenergic receptor (ADRB3) is associated with obesity, insulin resistance and earlier onset of type 2 diabetes mellitus. The aim of our study was evaluation of frequency of this ADRB3 polymorphism and his association with metabolic syndrome parameters and oxidative stress in postmenopausal women. MATERIAL AND METHODS: We performed the study among 94 women, aged 50-60, selected randomly from Wroclaw city population. Estimation of anthropometric parameters, densitometry (total body fat, android and gynoid deposits--using DPX(+) Lunar, USA device) and biochemical estimations such as lipid profile, glucose, insulin, estradiol and FSH serum level (using commercial kits) were carried out. Oxidative stress was estimated by measurement of thiobarbituric-reactive substances (TBARS) serum concentration, using Yagi method, on spectrofluorimeter Perkin-Elmer LS55. Blood for analysis was collected before, direct after and 6 h after the 30-minutes physical test using cycloergometer. ADRB3 genotyping was performed by PCR and mini-sequencing using ABI 310 sequencer (Applied Biosystems). RESULTS: The frequency of Trp(64)/Arg(64) genotype in investigated population was 15.8%. The Arg(64)/Arg(64) genotype had only one woman. Women bearing Trp(64)/Arg(64) genotype showed higher mean serum level of triglycerides and lower serum level of HDL-cholesterol in comparison to women bearing Trp(64)/Trp(64) genotype, however without statistical significance (p > 0.05) (respectively, means +/- SD for triglycerides: 140.3 +/- 64.1 vs. 113.9 +/- 56.2 mg/dl; and for HDL-cholesterol: 60.9 +/- 11.9 vs. 67.0 +/- 16.9 mg/dl). Both groups did not differ in any other investigated anthropometric nor biochemical parameter. CONCLUSIONS: 1. The Trp(64)/Arg(64) polymorphism of beta(3)-adrenergic receptor could be associated with lipid profile disorders observed in metabolic syndrome in postmenopausal women, however it should be explained basing on the study with more included subjects. 2. The Trp(64)/Arg(64 )polymorphism of beta(3)-adrenergic receptor has no influence on oxidative stress intensification after standardized physical effort in postmenopausal women.     

ACE insertion/deletion polymorphism and submaximal exercise hemodynamics in postmenopausal women.

We sought to determine whether the angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism is associated with submaximal exercise cardiovascular hemodynamics. Postmenopausal healthy women (20 sedentary, 20 physically active, 22 endurance athletes) had cardiac output (acetylene rebreathing) measured during 40, 60, and 80% VO(2 max) exercise. The interaction of ACE genotype and habitual physical activity (PA) level was significantly associated with submaximal exercise systolic blood pressure, with only sedentary women exhibiting differences among genotypes. No significant effects of ACE genotype or its interaction with PA levels was observed for submaximal exercise diastolic blood pressure. ACE genotype was significantly associated with submaximal exercise heart rate (HR) with ACE II having approximately 10 beats/min higher HR than ACE ID/DD genotype women. ACE genotype did not interact significantly with habitual PA level to associate with submaximal exercise HR. ACE genotype was not independently, but was interactively with habitual PA levels, associated with differences in submaximal exercise cardiac output and stroke volume. For cardiac output, ACE II genotype women athletes had ~25% greater cardiac output than ACE DD genotype women athletes, whereas for stroke volume genotype-dependent differences were observed in both the physically active and athletic women. ACE genotype was not significantly associated, either independently or interactively with habitual PA levels, with submaximal exercise total peripheral resistance or arteriovenous O(2) difference. Thus the common ACE locus polymorphic variation is associated with many submaximal exercise cardiovascular hemodynamic responses.     

Variable-length poly-C tract polymorphisms of the beta2-adrenergic receptor 3'-UTR alter expression and agonist regulation

Beta(2)-adrenergic receptors (beta(2)-AR) expressed on airway epithelial and smooth muscle cells regulate mucociliary clearance and relaxation and are the targets for beta-agonists in the treatment of obstructive lung disease. However, the clinical responses display extensive interindividual variability, which is not adequately explained by genetic variability in the 5'-flanking or coding region of the intronless beta(2)-AR gene. The nonsynonymous coding polymorphism most often associated with a bronchodilator phenotype (Arg16) is found within three haplotypes that differ by the number of Cs (11, 12, or 13) within a 3'-untranslated region (UTR) poly-C tract. To examine potential effects of this variability on receptor expression, BEAS-2B cells were transfected with constructs containing the beta(2)-AR (Arg16) coding sequence followed by its 3'-UTR with the various polymorphic poly-C tracts. beta(2)Arg16-11C had 25% lower mRNA expression and 33% lower beta(2)-AR protein expression compared with the other two haplotypes. Consistent with this lower steady-state expression, beta(2)Arg16-11C mRNA displayed more rapid and extensive degradation after actinomycin D treatment compared with beta(2)Arg16-12C and -13C. However, beta(2)Arg16-12C underwent 50% less downregulation of receptor expression during beta-agonist exposure compared with the other two haplotypes. Thus these haplotypes direct a potential low-response phenotype due to decreased steady-state receptor expression combined with wild-type agonist-promoted downregulation (beta(2)Arg16-11C) and a high-response phenotype due to increased baseline expression combined with decreased agonist-promoted downregulation (beta(2)Arg16-12C). This heterogeneity may contribute to the variability of clinical responses to beta-agonist, and genotyping to identify these 3'-UTR polymorphisms may improve predictive power within the context of beta(2)-AR haplotypes in pharmacogenetic studies.     

β2肾上腺素受体遗传多态性研究进展

β2肾上腺素受体（β2-adrenergic receptor,132-AR）对血管和支气管平滑肌的紧张性起着重要的调节作用,能介导心脏的正性变力和变时效应。近年来研究发现,人类β2-AR具有遗传多态性,而使受体表现出不同的生物学特性。本文主要对β2-AR的遗传多态性及遗传药理学的研究进展进行简要概述。     

Beta 1-adrenergic receptor polymorphisms confer differential function and predisposition to heart failure

Catecholamines stimulate cardiac contractility through beta(1)-adrenergic receptors (beta(1)-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased beta-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and G alpha(s) expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to beta-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to beta-receptor blockade.     

心脏β2—肾上腺素受体研究进展

随着受体的研究的蓬勃发展,对在心脏活动调节中起重要作用的肾上腺素受体的了解也更加深入。近年来的许多研究表明β2－肾上腺素受体不同亚型之间的信号转导及其介质的心脏反应有着很大的差异。本文扼要介绍了心脏β2－肾上腺素受体的最新研究进展,主要包括β2－肾上腺素受体中的混杂G蛋白偶联、信号转导局域化、固有活性及其与充血性心力衰竭的关系。     

Beta 3-adrenergic receptor is involved in feeding regulation in chicks

We examined whether the brain beta 3-adrenergic receptor (B3-AR) is involved in the feeding regulation of chicks. Intracerebroventricular (ICV) injection of BRL37344, a B3-AR agonist, reduced food intake of chicks under ad libitum, but not fasting, feeding conditions. The ICV injection of BRL37344 did not affect chick posture or locomotion activity suggesting that BRL37344 inhibited feeding without induction of sleep-like behavior as caused by norepinephrine. Furthermore, the rectal temperature increased following the ICV injection of BRL37344. Intraperitoneal administration of BRL37344 did not reduce food intake under ad libitum feeding condition. The present study demonstrated that the brain B3-AR is involved in the inhibition of feeding in chicks. We also suggested that activation of the brain affects the energy metabolism in chicks.     

Alterations in beta 1- and beta 2-adrenergic receptor density in the cerebellum of aging rats

Abstract: The densities of β¹, and β₂-adrenergic receptors were determined in homogenates of cerebral cortex and cerebellum of rats between 3 and 14 mo of age. No change in either receptor population occurred in the cortex during this period. In the cerebellum, a 20–25% decrease in the density of β₂, receptors and a 3509% increase in the density of β₁, receptors occurred. The increase in β₁ receptors in the cerebellum may be the result of a decrease in the function of the noradrenergic projections from the locus coeruleus which synapse on cerebellar Purkinje cells.     

Beta(2)-adrenergic receptors potentiate glucocorticoid receptor transactivation via G protein beta gamma-subunits and the phosphoinositide 3-kinase pathway

Glucocorticoid hormones influence manifold neuronal processes including learning, memory, and emotion via the glucocorticoid receptor (GR). Catecholamines further modulate these functions, although the underlying molecular mechanisms are poorly understood. Here, we show that epinephrine and norepinephrine potentiate ligand-dependent GR transactivation in a hippocampal cell line (HT22) via beta(2)-adrenergic receptors. This enhancement was strongest at low concentrations of glucocorticoids and was accompanied by increased GR binding to a glucocorticoid-responsive element (GRE). beta(2)-Adrenergic receptor-mediated GR enhancement was relayed via G protein beta gamma-subunits, insensitive to pertussis toxin and independent of protein kinase A (PKA). In contrast, the catecholamine-evoked GR enhancement was strongly reduced by wortmannin, suggesting a critical role for phosphoinositide 3-kinase (PI3-K). In agreement, epinephrine directly activated PI3-K in vivo. Similarly, stimulation of tyrosine kinase receptors coupled to PI3-K activation, e.g. receptors for insulin-like growth factor I (IGF-I) or fibroblast growth factor (FGF), increased GR transactivation. Further analysis indicated that G protein-coupled receptor (GPCR) and tyrosine kinase receptor signals converge on PI3-K through separate mechanisms. Blockade of GR enhancement by wortmannin was partially overcome by expression of the downstream-acting protein kinase B (PKB/Akt). Collectively, our findings demonstrate that GPCRs can regulate GR transactivation by stimulating PI3-K. This novel cross-talk may provide new insights into the molecular processes of learning and memory and the treatment of stress-related disorders.     

Mammalian beta 1- and beta 2-adrenergic receptors. Immunological and structural comparisons

Beta 1- and beta 2-adrenergic receptors, pharmacologically distinct proteins, have been reported to be structurally dissimilar. In the present study three techniques were employed to compare the nature of mammalian beta 1- and beta 2-adrenergic receptors. Antibodies against each of the receptor subtypes were raised separately. Polyclonal antisera against beta 1-receptors of rat fat cells were raised in mice, and antisera against beta 2-receptors of guinea pig lung were raised in rabbits. Receptors purified from rat fat cells (beta 1-), S49 mouse lymphoma cells (beta 2-), and rat liver (beta 2-) were probed with these antisera. Each anti-receptor antisera demonstrated the ability to immunoprecipitate purified receptors of both beta 1- and beta 2- subtypes. The mobility of beta-receptors subjected to polyacrylamide gel electrophoresis was probed using antireceptor antibodies and nitrocellulose blots of the gels. Fat cell beta 1-adrenergic receptors display Mr = 67,000 under reducing conditions and Mr = 54,000 under nonreducing conditions, as previously reported (Moxham, C. P., and Malbon, C. C. (1985) Biochemistry 24, 6072-6077). Both beta 1- and beta 2-receptors displayed this same shift in electrophoretic mobility observed in the presence as compared to the absence of disulfide bridge-reducing agents, as detected both by autoradiography of the radiolabeled receptors and by immunoblotting of native receptors. Finally, isoelectric focusing of purified radioiodinated beta 1- and beta 2-adrenergic receptors revealed identical isoelectric points. These data are the first to provide analyses of immunological, structural, and biochemical features of beta 1- and beta 2-subtypes in tandem and underscore the structural similarities that exist between these pharmacologically distinct receptors.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.     

Myocardial beta1-adrenergic receptor polymorphisms affect functional recovery after ischemic injury

Association studies suggest beta(1)-adrenergic receptor (beta(1)-AR) polymorphisms are disease modifiers in heart failure. The Arg389 variant has increased coupling to G(s) in transfected cells and evokes enhanced ventricular function in transgenic mice. Here, we assessed the differential effects of the human Gly389 and Arg389 beta(1)-AR polymorphisms on myocardial recovery after ischemic injury. Function was studied in transgenic mice with cardiac-specific expression of either human Gly389 or Arg389 beta(1)-AR at baseline and after 20 min of ex vivo ischemia and reperfusion (I/R). In 3-mo-old mice of either genotype, there was poor recovery after I/R (approximately 38% vs. approximately 68% for nontransgenic). Paradoxically, at 6 mo of age, functional recovery remained severely depressed in Gly389 hearts (approximately 32%) but was similar to nontransgenic for Arg389 hearts (approximately 60%). In Arg389 hearts, agonist-promoted adenylyl cyclase activities were depressed by approximately 35% at 6 mo of age, and G protein-coupled receptor kinase (GRK) activity was increased by approximately twofold compared with Gly389. Furthermore, I/R evoked an approximately threefold increase in ERK2 phosphorylation in Arg389 but an approximately twofold decrease in Gly389 hearts. Individually, these changes have been shown to mitigate I/R injury; thus the Arg389-beta(1)-AR uniquely evokes specialized pathways that act to protect against I/R injury. The improved recovery of function after I/R in Arg389 hearts relative to Gly389 appears to be due to an adaptive multimechanism program with allele-specific alterations in receptor signaling, GRK activity, and ERK2. Thus genetic variation of the human beta(1)-AR may play a role in cardiac functional recovery after ischemic injury.     

Arg16/Gly beta2-adrenergic receptor polymorphism alters the cardiac output response to isometric exercise.

Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic-receptor polymorphism have 1) greater forearm beta2-receptor mediated vasodilation and 2) a higher heart rate (HR) response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the higher HR response in Gly16 subjects serves to maintain the pressor response [increased cardiac output (CO)] in the setting of augmented peripheral vasodilation to endogenous catecholamines, we measured continuous HR (ECG), arterial pressure (Finapres), and CO (transthoracic echocardiography) during isometric, 40% submaximal handgrip to fatigue in healthy subjects homozygous for Gly (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg (n = 17, age 30 +/- 1.6, 11 women). Resting data were similar between groups. Handgrip produced similar increases in arterial pressure and venous norepinephrine and epinephrine concentrations; however, HR increased more in the Gly group (60.1 +/- 4.3% increase from baseline vs. 45.5 +/- 3.9%, P = 0.03), and this caused CO to be higher (Gly: 7.6 +/- 0.3 l/m vs. Arg: 6.5 +/- 0.3 l/m, P = 0.03), whereas the decrease in systemic vascular resistance in the Gly group did not reach significance (P = 0.09). We conclude that Gly16 homozygotes generate a higher CO to maintain the pressor response to handgrip. The influence of polymorphic variants in the beta2-adrenergic receptor gene on the cardiovascular response to sympathoexcitation may have important implications in the development of hypertension and heart failure.     

beta-arrestin-biased agonism at the beta2-adrenergic receptor

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.