Homo- and hetero-oligomerization of β2-adrenergic receptor in receptor trafficking,signaling pathways and receptor pharmacology

The β₂-adrenergic receptor (β₂AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β₂AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β₂AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β₂AR oligomerization with focus on the implications of the receptor–receptor interactions to β₂AR trafficking, pharmacology and downstream signal transduction pathways.     

Agonist-directed desensitization of the β2-adrenergic receptor

The β(2)-adrenergic receptor (β(2)AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β(2)AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β(2)AR desensitization at the whole cell level.     

Connectivity and information transfer in flow networks: Two magic numbers in ecology?

An ecosystem can be visualized as a graph of certain preassigned trophic compartments; these nodes are then mutually connected through the internal exchanges of material and energy. The mathematical theory of information can be applied to such a graph in order to define two relevant indices: a measure of connectivity (the entropy H of the connections) and a measure of the degree of the “energetic” specialization (the internal transfer of informationI). The computation of these indices in stationary real cases suggests that the observed complexity of ecosystems is conditioned by two competing effects. The first can be interpreted as a “thermodynamical” principle related to the unavoidable irreversibility taking place inside the system, whereas the second can be taken as a “biological” principle concerned with the selection of some particular interactions: those which maximize the information circulating between the compartments.     

Multiple ligand-specific conformations of the β2-adrenergic receptor

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.     

Analysis of L-DOPA and droxidopa binding to human β2-adrenergic receptor

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β₂-adrenergic receptor (β₂AR). The 3.2 Å x-ray crystal structure of human β₂AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β₂AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β₂AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β₂AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β₂AR-ligand complexes performed here.     

Biological maturation and β-adrenergic effectors: development of β-adrenergic receptors in rabbit heart

Summary The -adrenergic receptor, transduction processes and catalytic activity of the adenylate cyclase enzyme complex have been investigated in rabbit heart at different stages of biological maturation. The binding of [³H]-dihydroalprenolol to a washed membrane preparation isolated from rabbit ventricular muscle was used to characterize -adrenergic receptors. Significant age-related differences were noted in -receptor affinity (K_d) and density (RD) of neonatal and adult animals; the adult K_d was 3.7-fold greater and the RD 2-fold higher than the neonates. No significant differences in these parameters were detected among the 27-day old fetus and the 1- and 7-day old neonates. Age-dependent differences in agonist isoproterenol affinity for the receptor were not observed in contrast to the significant changes in antagonist (DHA) affinity.Age-related changes in receptor affinity were also quantitated by determining the inhibitory potency of alprenolol on isoproterenol stimulated adenylate cyclase enzyme activity. A decreased affinity of the -adrenergic receptor for alprenolol in the adult heart was indicated by a 3.7-fold greater K_i for the adult than the 1-day old neonate. Ontogenic variations in the coupling efficiency between the receptor and catalytic components of the adenylate cyclase complex were also evaluated. The K_d of the -adrenergic receptor for isoproterenol and the EC50 for adenylate cyclase stimulation were determined under similar conditions. The corresponding coupling index (K_d/EC50) was found to be 2.4-fold greater in the 1-day old neonate than adult, suggesting that for a given percentage increase in adenylate cyclase activity, a lower percentage of -adrenergic receptor sites need be occupied in the neonate. These data extend previous studies (29) and indicate all components of the rabbit heart adenylate cyclase enzyme complex (i.e., the -adrenergic receptor, the GTP-dependent transduction event, and the catalytic subunit) exhibit significant developmental changes.     

Age-related up-regulation of β3-adrenergic receptor in heart-failure rats

Stimulation of β1- and β2-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the β3-AR is also expressed in the heart and that its stimulation leads to negative inotropic effects. The aim of this study was to investigate the expression of β3-AR in age-related heart-failure rats and its relevance to left ventricular dysfunction. Aging male Wistar rats were divided into young and aging groups according to age, and each group included sham-operation and heart-failure subgroups. Left ventricular end-diastolic pressure (LVEDP) and the ratio of left ventricular weight to body weight (LV/BW) were significantly higher for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). However, the left ventricular end-systolic pressure (LVESP) and the maximal rate of rise or fall of left ventricular pressure were all significantly lower for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). β3-AR protein levels increased significantly when heart failure worsened in aging rats. These results suggest that β3-AR expression in age-related heart-failure rats and left ventricular function were highly correlated.     

Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective β2-adrenergic receptor agonists

Purpose: To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the β₂-adrenergic receptor. Results: Out of 21 computationally designed structures 6 compounds were synthesized and characterized for β₂-AR binding affinities, subtype selectivities and functional activities. Conclusion: the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K_iβ₂-AR = 0.28 μm, K_iβ₁-AR/K_iβ₂-AR = 573, EC_50cAMP = 3.9 nm, EC_50cardio = 16 nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the β2-adrenergic receptor: Part 6

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human β₃-adrenergic receptor (AR) agonists. Result of structure–activity relationships (SAR) and cassette-dosing evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray structure of the β₂-adrenergic receptor.     

Nuclear GPCRs in cardiomyocytes: an insider's view of β-adrenergic receptor signaling

In recent years, we have come to appreciate the complexity of G protein-coupled receptor signaling in general and β-adrenergic receptor (β-AR) signaling in particular. Starting originally from three β-AR subtypes expressed in cardiomyocytes with relatively simple, linear signaling cascades, it is now clear that there are large receptor-based networks which provide a rich and diverse set of responses depending on their complement of signaling partners and the physiological state. More recently, it has become clear that subcellular localization of these signaling complexes also enriches the diversity of phenotypic outcomes. Here, we review our understanding of the signaling repertoire controlled by nuclear β-AR subtypes as well our understanding of the novel roles for G proteins themselves in the nucleus, with a special focus, where possible, on their effects in cardiomyocytes. Finally, we discuss the potential pathological implications of alterations in nuclear β-AR signaling.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Antiangiogenic effects of β2 -adrenergic receptor blockade in a mouse model of oxygen-induced retinopathy

Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity. In mice with OIR, beta-adrenergic receptor (β-AR) blockade with propranolol has been shown to ameliorate different aspects of retinal dysfunction in response to hypoxia. In the present study, we used the OIR model to investigate the role of distinct β-ARs on retinal proangiogenic factors, pathogenic neovascularization and electroretinographic responses. Our results demonstrate that β(2) -AR blockade with ICI 118,551 decreases retinal levels of proangiogenic factors and reduces pathogenic neovascularization, whereas β(1) - and β(3) -AR antagonists do not. Determination of retinal protein kinase A activity is indicative of the fact that β-AR blockers are indeed effective at the receptor level. In addition, the specificity of ICI 118,551 on retinal angiogenesis has been demonstrated by the finding that in mouse retinal explants, β(2) -AR silencing prevents ICI 118,551 effects on hypoxia-induced vascular endothelial growth factor accumulation. In OIR mice, ICI 118,551 is effective in increasing electroretinographic responses suggesting that activation of β(2) -ARs constitutes an important part of the retinal response to hypoxia. Lastly, immunohistochemical studies demonstrate that β(2) -ARs are localized to several retinal cells, particularly to Müller cells suggesting the possibility that β(2) -ARs play a role in regulating vascular endothelial growth factor production by these cells. The present results suggest that pathogenic angiogenesis, a key change in many hypoxic/ischemic vision-threatening retinal diseases, depends at least in part on β(2) -AR activity and indicate that β(2) -AR blockade can be effective against retinal angiogenesis.     

α2 and β-adrenergic stimulation of corticosterone secretion in rats

Bilateral injection of 6-hydroxydopamine into the medial forebrain bundle (MFB) significantly decreased monoamine concentrations in the hypothalamus. The noradrenaline and serotonin content of the paraventricular nucleus (PVN) was also significantly reduced. These drastic decreases in neurotransmitter concentration did not alter basal secretion of corticosterone. Isoproterenol. a -adrenoceptor agonist (1 mg/kg, i.p.), significantly stimulated corticosterone release in saline and MFB lesioned rats. This stimulation did not differ significantly between the two groups. Clonidine, an ₂-adreceptor agonist, injected either intraperitoneally or intracerebrally just dorsal to the PVN, caused a dose-dependent increase in corticosterone secretion. The stimulation of corticosterone release by clonidine (250 g/kg, i.p.) was antagonised by the selective ₂-adreceptors antagonist, yohimbine (1 mg/kg, i.p.) and significantly reduced by the MFB lesion. These results suggest that corticosterone secretion is stimulated by activation of ₂-adreceptors which occur on noradrenergic nerve terminals in the PVN.     

Association study of the β2-adrenergic receptor gene polymorphisms and hypertension in the Northern Han Chinese

Background

The β2-adrenergic receptor (ADRB2) gene has been widely researched as a candidate gene for essential hypertension (EH), but no consensus has been reached in different ethnicities. The aim of the present study was to evaluate the possible association between the ADRB2 gene polymorphisms and the EH risk in the Northern Han Chinese population.

Methodology/Principal Findings

This study included 747 hypertensive subjects and 390 healthy volunteers as control subjects in the Northern Han Chinese. Genotyping was performed to identify the C-47T, A46G and C79G polymorphisms of the ADRB2 gene. G allelic frequency of A46G polymorphism was significantly higher in hypertensive subjects (P = 0.011, OR = 1.287, 95%CI [1.059–1.565]) than that in controls. Significant association could also be found in dominant genetic model (GG+AG vs. AA, P = 0.006, OR = 1.497, 95%CI [1.121–1.998]), in homozygote comparison (GG vs. AA, P = 0.025, OR = 1.568, 95%CI [1.059–2.322]), and in additive genetic model (GG vs. AG vs. AA, P = 0.012, OR = 1.282, 95%CI [1.056–1.555]). Subgroup analyses performed by gender suggested that this association could be found in male, but not in female. Stratification analyses by obesity showed that A46G polymorphism was related to the prevalence of hypertension in the obese population (GG vs. AG vs. AA, P<0.001, OR = 1.645, 95%CI [1.258–2.151]). Significant interaction was found between A46G genotypes and body mass index on EH risk. No significant association could be found between C-47T or C79G polymorphism and EH risk. Linkage disequilibrium was detected between the C-47T, A46G and C79G polymorphisms. Haplotype analyses observed that the T-47-A46-C79 haplotype was a protective haplotype for EH, while the T-47-G46-C79 haplotype increased the risk.

Conclusions/Significances

We revealed that the ADRB2 A46G polymorphism might increase the risk for EH in the Northern Han Chinese population.