β2AR Function Research Articles

Microtubule-Mediated Regulation of β2AR Translation and Function in Failing Hearts.

β1AR (beta-1 adrenergic receptor) and β2AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac β-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible β-AR translation sites in cardiomyocytes. The mechanism by which β-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes. β1AR and β2AR mRNAs show differential localization in cardiomyocytes, with β1AR found in the perinuclear region and β2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β2AR transcripts toward the perinuclear region. The close proximity between β2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β1AR and β2AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to β-AR mRNA redistribution and impaired β2AR function in failing hearts. Asymmetrical microtubule-dependent trafficking dictates differential β1AR and β2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted β2AR-mediated cyclic adenosine monophosphate signaling.

Fingerprinting heterocellular β-adrenoceptor functional expression in the brain using agonist activity profiles.

Noradrenergic projections from the brainstem locus coeruleus drive arousal, attentiveness, mood, and memory, but specific adrenoceptor (AR) function across the varied brain cell types has not been extensively characterized, especially with agonists. This study reports a pharmacological analysis of brain AR function, offering insights for innovative therapeutic interventions that might serve to compensate for locus coeruleus decline, known to develop in the earliest phases of neurodegenerative diseases. First, β-AR agonist activities were measured in recombinant cell systems and compared with those of isoprenaline to generate Δlog(Emax/EC50) values, system-independent metrics of agonist activity, that, in turn, provide receptor subtype fingerprints. These fingerprints were then used to assess receptor subtype expression across human brain cell systems and compared with Δlog(Emax/EC50) values arising from β-arrestin activation or measurements of cAMP response desensitization to assess the possibility of ligand bias among β-AR agonists. Agonist activity profiles were confirmed to be system-independent and, in particular, revealed β2-AR functional expression across several human brain cell types. Broad β2-AR function observed is consistent with noradrenergic tone arising from the locus coeruleus exerting heterocellular neuroexcitatory and homeostatic influence. Notably, Δlog(Emax/EC50) measurements suggest that tested β-AR agonists do not show ligand bias as it pertains to homologous receptor desensitization in the system examined. Δlog(Emax/EC50) agonist fingerprinting is a powerful means of assessing receptor subtype expression regardless of receptor expression levels or assay readout, and the method may be applicable to future use for novel ligands and tissues expressing any receptor with available reference agonists.

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor β2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The β2-adrenergic receptor (β2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. β2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length β2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length β2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length β2ARs with distinct modification patterns, including a full-length β2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length β2AR receptors, we found that different phosphorylation patterns mediate different interactions with β-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between β2AR and β-arrestin1. Access to full-length β2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of β2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

Differential Modulation of β2AR Mediated cAMP/PKA/CREB Signaling Cascade in Normal versus Asthmatic Airway Epithelial Cells

Nearly 25 million people in the U.S. suffer from asthma. While there is no panacea for asthma, symptomatic relief is achieved by the current therapeutic interventions, including β2-adrenergic receptor (β2AR) agonists. Chronic usage of β2AR agonists can have serious implications like airway hyper-responsiveness and receptor desensitization, and along with unknown mechanisms, can ultimately culminate into tachyphylaxis to the response. Previously, our laboratory has extensively explored the critical role of reactive oxygen species (ROS) in modulating β2AR function. To begin to assess the physiological role of the β2AR-ROS linkage in asthma, here, we assessed Isoproterenol (ISO)-induced β2AR mediated Gαs/cAMP/CREB pathway in small airway epithelial cells from asthma patients (SAEC-A) compared to those from non-diseased control patients (SAEC-N). Given the importance of β2AR mediated cAMP production and its role in airway bronchodilation, as well as tachyphylaxis that occurs upon chronic use of β2AR agonists, our results may shed light on the role of oxidants in altering β2AR responses in asthma. To assess cAMP levels, cells were analyzed using highly sensitive luciferase-based biosensor. Briefly, SAEC were transfected with GloSensorTM -22F cAMP plasmid (8μg) using Lipofectamine3000. Luminescence was measured following treated with the ISO. To assess the effects of downstream cAMP-mediated transcription, cells were stimulated with H2O, Forskolin (FSK) (10 μM) & ISO (100μM) for 20 min and lysed. Lysates were resolved using SDS-PAGE and immunoprobed for phospho-cAMP response element-binding protein (pCREB), total CREB and β-actin. Similarly, SAEC-N/A lysates were assessed for Adenylyl Cyclase (AC-5/6) and Phosphodiesterase (PDE4) by immunoblot. β2AR agonism with ISO significantly increased cAMP production in SAEC from asthma patients compared to non-diseased control. In spite of elevated cAMP production in asthmatic SAEC, both ISO and FSK were able to induce significant increase in CREB phosphorylation, leading to heightened pCREB/CREB response in SAEC-N as compared to SAEC-A. ISO-mediated increased cAMP response in asthmatic SAEC compared to non-diseased control, were linked with physiologically altered expression of key cAMP mediators, specifically, heightened basal expression of AC-5/6 and PDE4, in asthmatic SAEC compared to SAEC-N. ISO mediated increased cAMP production in asthmatic SAEC indicated altered agonist modulated β2AR downstream signaling in diseased versus non-diseased SAEC. Further, heightened basal expression of AC-5/6 in asthmatic SAEC supports the above mentioned consequence. In addition, elevated CREB phosphorylation in non-diseased SAEC suggests alterations of β2AR-mediated PKA/CREB signaling in asthma. Future results will probe the effects of elevated ROS in these signal cascades.

Aldosterone And Osteopontin Promote Cardiac Fibrosis Via GRK2‐dependent β2‐Adrenergic Receptor Dysfunction

BackgroundCardiac β2‐adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β2AR is a Gs protein‐coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3′, 5′‐adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac1/2). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro‐inflammatory and pro‐fibrotic cytokine, including in the heart. OPN expression is known to be induced by the cardiotoxic hormone aldosterone in various tissues and OPN has been reported to dysregulate β2AR signaling and function in bone cells. Thus, we hypothesized here that aldosterone upregulates OPN in the heart in order to perturb β2AR anti‐fibrotic function.MethodsWe used the rat cardiomyoblast cell line H9c2 and human cardiac fibroblasts (HCFs). We measured OPN levels and fibrosis markers via real time PCR and β2AR function via cAMP accumulation studies and co‐immunoprecipitation/western blotting for several downstream signaling mediators. We also deleted OPN via CRISPR/Cas9 to examine its role in cardiac β2AR function and fibrosis.ResultsAldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyocytes. This is prevented by β2AR activation with salbutamol (albuterol), which stimulates GPCR‐kinase (GRK)‐5 to phosphorylate the cardiac MR and inhibit its transcriptional activity. Importantly, CRISPR/Cas9‐mediated OPN gene deletion enhances β2AR‐dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1 protein levels. The functional result of this is that β2AR’s inhibition of transforming growth factor (TGF)‐β‐dependent fibrosis in H9c2 cardiomyocytes is robustly augmented. In addition, pharmacological inhibition of GRK2, the major GRK that phosphorylates and desensitizes the cardiac β2AR, with Cmpd101 or OPN CRIPSR/Cas9‐mediated knockout completely abrogates isoproterenol (agonist)‐induced β2AR functional desensitization in HCFs (Isoproterenol’s ΔEC50: 94±16 μM in control HCFs, p<0.05, n=4; 6±7.2 μM in Cmpd101‐treated HCFs, Not significant at p=0.05, n=4; 5.3+6.1 μM in OPN knockout HCFs, Not significant at p=0.05, n=4). Mechanistically, OPN dysregulates the β2AR via enhancement of GRK2‐mediated functional desensitization, since OPN forms a multi‐protein complex with GRK2 and GRK2‐interacting protein (GIT)‐1 in HCFs, thereby facilitating GRK2 plasma membrane recruitment. This reduces β2AR’s anti‐fibrotic cAMP signaling in the heart.ConclusionsWe have uncovered a direct inhibitory effect of OPN on cardiac β2AR’s anti‐fibrotic signaling: facilitation of GRK2‐mediated receptor desensitization. Since aldosterone/MR upregulates OPN, a combination of an MR blocker with OPN or GRK2 blockade could be of value in cardiac fibrosis treatment.Support or Funding Information1) American Heart Association (A.L.)2) NSU President’s Faculty Research & Development Grant (A.L.)

Interactive Effects of Zinc and Zilpaterol Hydrochloride on Bovine <i>β</i>-Adrenergic Receptors

The objective of this study was to determine if the addition of zinc (Zn) in combination with zilpaterol HCL (ZH) affected the interaction of ZH with the beta2-adrenergic receptor (β-AR) by altering cAMP production, gene expression, and protein abundance in cultured skeletal muscle cells. Cultures of muscle bovine satellite cells were established and treated at 120 h with: 1) 0 μM Zn/zilpaterol hydrochloride (ZH; CON); 2) 0 μM Zn/10 μM ZH (ZH); 3) 1 μM Zn from Zn chloride/0 μM ZH (Zn); 4) 1 μM Zn from Zn chloride/10 μM ZH (ZN/ZH) in differentiation media for an additional 0, 6, 24, 48 and 96 h. Protein and mRNA were isolated and quantified at 24 and 96 h, and cAMP was measured at 0, 6, 24, 48 and 96 h. At 0, 24, 48 and 96 h, no differences (P > 0.05) were detected in cAMP production. At 6 h, Zn cells had the greatest concentration of cAMP (P P > 0.05) were detected in mRNA abundance at 24 h. At 96 h, 0 μM Zn/10 μM ZH cells had an increased abundance of myosin heavy chain (MHC)-I mRNA (P P P P > 0.05) were detected in the protein abundance of β1AR and the β2AR. These results indicated Zn and ZH in combination did not have an additive effect on β2-AR function as indicated by cAMP concentrations.

Neurogenic effect of exercise via the thioredoxin-1/ extracellular regulated kinase/β-catenin signaling pathway mediated by β2-adrenergic receptors in chronically stressed dentate gyrus.

Chronic stress is a precipitating factor for depression, whereas exercise is beneficial for both the mood and cognitive process. The current study demonstrates the anti-depressive effects of regular exercise and the mechanisms linked to hippocampal neurogenesis. Mice were subjected to 14 consecutive days of restraint, followed by 3 weeks of treadmill running, and were then subjected to behavioral tests that included the forced swimming and Y-maze tests. Protein levels were assessed using western blot analysis and newborn cells were detected using 5-bromo-2'-deoxyuridine (BrdU). Three weeks of treadmill running ameliorated the behavioral depression caused by 14 days of continuous restraint stress. The exercise regimen enhanced BrdU-labeled cells and class III β-tubulin levels in the hippocampal dentate gyrus, as well as those of thioredoxin-1 (TRX-1) and synaptosomal β2-adrenergic receptors (β2-AR) under stress. In vitro experiments involving treatment with recombinant human TRX-1 (rhTRX-1) augmented the levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2), nuclear β-catenin, and proliferating cell nuclear antigens, which were previously inhibited by U0216 and FH535 (inhibitors of ERK1/2 and β-catenin/T cell factor-mediated transcription, respectively). The hippocampal neurogenesis elicited by a 7-day exercise regimen was abolished by a selective inhibitor of β2-AR, butoxamine. These results suggest that TRX-1-mediated hippocampal neurogenesis by β2-AR function is a potential mechanism underlying the psychotropic effect of exercise.

Abstract 469: Targeting Beta Adrenergic Receptor Resensitization Aids in Preservation of Receptor Function

Beta adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and is an integral contributor to heart failure. We have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also generated a small molecule from our in silico predictions that could target the interface of I2PP2A and PP2A binding to find that disruption of the PP2A/I2PP2A interaction underlies receptor function. We will use this small molecule to look at preservation of βAR function and amelioration of cardiac function. To test whether resensitization is altered in heart failure we used plasma membrane and endosomal fractions from non-failing and paired pre- and post-LVAD samples to show PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase activity as a measure of recovery in βAR function. Our studies showed that endosomal fractions from human heart failure samples had elevated PI3K activity associated with reduced PP2A activity supporting the idea that βAR resensitization is inhibited in human heart failure samples. Since human heart failure samples have inhibited resensitization we tested the underlying mechanisms regulating βAR resensitization. Thus ongoing studies suggest that targeting the resensitization of βAR could provide beneficial cardiac remodeling in conditions of chronic mechanical overload and will be further discussed.

Abstract 128: Impaired Beta Adrenergic Receptor Resensitization Contributes to Human Heart Failture

Beta adrenergic receptors (βARs) are one of the most powerful regulators of cardiac function, and βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization by G-protein coupled receptor kinase phosphorylation leading to βAR internalization. Desensitized βARs are resensitized by dephosphorylation in the endosomes by PP2A; however, less is known about role of resensitization in heart failure. Although we have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on in silico predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also tested whether resensitization as a mechanism is altered in endstage human heart failure samples. Since resensitization occurs in endosomes, plasma membranes and endosomes were isolated from human heart samples and assessed for PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase (AC) activity as a measure of βAR function. Significant PI3Kγ activity was observed to increase in heart failure samples while AC activity was reduced in plasma membranes. Consistent with the reduced recovery in βAR function in heart failure samples, significant β2AR phosphorylation was observed in the endosomes and plasma membranes of human heart failure samples compared to non-failing. Correspondingly, we observed significant reduction in endosomal PP2A activity in heart failure samples versus controls suggesting inhibited resensitization occurs in human heart failure. Together our studies suggest that resensitization is inhibited in human heart failure and targeting I2PP2A may provide preservation of receptor function and beneficial cardiac remodeling.

IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism

β2-Adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of β2-agonists is well studied, type 2 inflammation could also affect β2AR function. We sought to evaluate the effect of the type 2 cytokine IL-13 on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2). HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and β2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2. Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients. IL-13 treatment of HAECs leads to β2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) β2ARs, suggesting that the beneficial effects of β2-agonists could be blunted in patients with type 2 associated asthma.

Quantification of beta adrenergic receptor subtypes in beta-arrestin knockout mouse airways.

In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*♦

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β2AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β2AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β2AR and provides valuable tools for the study of β2AR function.

Targeting α- and β-Adrenergic Receptors Differentially Shifts Th1, Th2, and Inflammatory Cytokine Profiles in Immune Organs to Attenuate Adjuvant Arthritis.

The sympathetic nervous system (SNS) regulates host defense responses and restores homeostasis. SNS-immune regulation is altered in rheumatoid arthritis (RA) and rodent models of RA, characterized by nerve remodeling in immune organs and defective adrenergic receptor (AR) signaling to immune cell targets. The SNS typically promotes or suppresses inflammation via α- and β2-AR activation, respectively, and indirectly drives humoral immunity by blocking Th1 cytokine secretion. Here, we investigate how β2-AR stimulation and/or α-AR blockade at disease onset affects disease pathology and cytokine profiles in relevant immune organs from male Lewis rats with adjuvant-induced arthritis (AA). Rats challenged to induce AA were treated with terbutaline (TERB), a β2-AR agonist (600 μg/kg/day) and/or phentolamine (PHEN), an α-AR antagonist (5.0 mg/kg/day) or vehicle from disease onset through severe disease. We report that in spleen, mesenteric (MLN) and draining lymph node (DLN) cells, TERB reduces proliferation, an effect independent of IL-2. TERB also fails to shift T helper (Th) cytokines from a Th1 to Th2 profile in spleen and MLN (no effect on IFN-γ) and DLN (greater IFN-γ) cells. In splenocytes, TERB, PHEN, and co-treatment (PT) promotes an anti-inflammatory profile (greater IL-10) and lowers TNF-α (PT only). In DLN cells, drug treatments do not affect inflammatory profiles, except PT, which raised IL-10. In MLN cells, TERB or PHEN lowers MLN cell secretion of TNF-α or IL-10, respectively. Collectively, our findings indicate disrupted β2-AR, but not α-AR signaling in AA. Aberrant β2-AR signaling consequently derails the sympathetic regulation of lymphocyte expansion, Th cell differentiation, and inflammation in the spleen, DLNs and MLs that is required for immune system homeostasis. Importantly, this study provides potential mechanisms through which reestablished balance between α- and β2-AR function in the immune system ameliorates inflammation and joint destruction in AA.

Agonist-induced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o− cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Structural Insights into the Dynamic Process of G Protein Coupled Receptor Activation

Research in my lab is directed at understanding the structural basis for the functional properties of G protein coupled receptors (GPCRs), which constitute the largest family of membrane proteins in the human genome. GPCRs conduct the majority of transmembrane responses to hormones and neurotransmitters, and mediate the senses of sight, smell and taste. The β2AR adrenoceptor (β2AR) is a prototypical Family A GPCR that mediates physiologic responses to adrenaline and noradrenaline. It regulates the activity of several distinct signaling pathways through both G protein dependent and G protein independent mechanisms. Like many GPCRs that respond to hormones and neurotransmitters, the β2AR exhibits modest basal activity in the absence of an agonist. This activity can be modulated by a spectrum of synthetic ligands ranging from inverse agonists, which suppress basal activity, to full agonists. We have obtained three-dimensional structures of the β2AR in inactive and active conformations, as well as a structure of the β2AR in complex with the G protein Gs. We have also used fluorescence, EPR and NMR spectroscopy to study the dynamic properties of the receptor, and to map ligand-specific conformational changes. I will discuss what we these studies have taught us about the structural basis of β2AR function.

Transient β2-Adrenoceptor Activation Confers Pregnancy Loss by Disrupting Embryo Spacing at Implantation

Pregnancy loss is a serious social and medical issue, with one important cause associated with aberrant embryo implantation during early pregnancy. However, whether and how the process of embryo implantation is affected by environmental factors such as stress-induced sympathetic activation remained elusive. Here we report an unexpected, transient effect of β2-adrenoreceptor (β2-AR) activation (day 4 postcoitus) in disrupting embryo spacing at implantation, leading to substantially increased midterm pregnancy loss. The abnormal embryo spacing could be prevented by pretreatment of β2-AR antagonist or genetic ablation of β-AR. Similar β2-AR activation at day 5 postcoitus, when implantation sites have been established, did not affect embryo spacing or pregnancy outcome, indicating that the adverse effect of β2-AR activation is limited to the preimplantation period before embryo attachment. In vitro and in vivo studies demonstrated that the transient β2-AR activation abolished normal preimplantation uterine contractility without adversely affecting blastocyst quality. The contractility inhibition is mediated by activation of the cAMP-PKA pathway and accompanied by specific down-regulation of lpa3, a gene previously found to be critical for uterine contraction and embryo spacing. These results indicated that normal uterine contraction-mediated correct intrauterine embryo distribution is crucial for successful ongoing pregnancy. Abnormal β2-AR activation at early pregnancy provided a molecular clue in explaining how maternal stress at early stages could adversely affect the pregnancy outcome.

In vitro hypoxia impairs β2-adrenergic receptor signaling in primary rat alveolar epithelial cells

Hypoxia inhibits beta(2)-adrenergic receptor (beta(2)-AR) signaling in a variety of tissues, but effects in alveolar epithelium are unclear. We therefore examined the effect of 24 h of hypoxia on beta(2)-AR function in primary rat alveolar epithelial [alveolar type II (ATII)] cells. ATII cells were isolated, cultured to confluence, and incubated in normoxia or hypoxia (3% O(2)) for 24 h. Hypoxia decreased maximal terbutaline-stimulated cAMP production by 37%; potency of terbutaline was not affected. Reoxygenation (3 h) reversed this effect. Density of beta(2)-AR assessed by (-)-[(125)I]iodocyanopindolol binding was decreased in hypoxia (-22%). Hypoxia did not affect terbutaline binding affinity to beta(2)-AR. Hypoxia decreased G(s) protein levels by 27%, whereas no change was observed in G(i1/2), G(i3), and Gbeta subunits. Forskolin-stimulated cAMP production was not inhibited by hypoxia. Pertussis toxin (PTX; 0.5 microg/ml, 2 h), an inhibitor of G(i/o) proteins, restored terbutaline-stimulated cAMP production of hypoxic ATII cells to normoxic control values. Cholera toxin (CTX)-stimulated G(s) protein activity did not change in hypoxia. Hypoxia increased the sensitivity of beta(2)-AR to desensitization. These results indicate that despite the decrease in G(s) protein level G(s) protein was still functional and that hypoxia impairs beta(2)-AR signaling due to an increased activity of G(i/o) proteins.

A polymorphism of G-protein coupled receptor kinase5 alters agonist-promoted desensitization of β2-adrenergic receptors

Beta-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of beta2-adrenergic (beta2AR) signaling genes. beta2AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for beta-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared with GRK5-Gln41. Consistent with this functional effect, agonist-promoted beta2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated beta2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of beta2AR during persistent agonist exposure, and thus may contribute to beta-agonist variability in asthma treatment of African-Americans.

Caveolin‐1 knockout alters β‐adrenoceptor (β‐AR) function in mouse small intestine

β-ARs transduce signals intracellularly via G-proteins. β2-ARs localize in caveolae and co-immunoprecipitate with caveolin-3 in cardiac myocytes. Cholesterol depletion disrupts caveolae and alters both β2-AR function and localization, suggesting that caveolins regulate these receptors. β-AR activation in mouse small intestine relaxes smooth muscle, mainly via β3, but also via β1 and β2-ARs. Here we examined the effects of caveolin-1 (cav1) knockout on β-AR function in mouse small intestine in vitro. In tissue segments contracted with carbachol, isoprenaline (iPr) induced relaxation. The iPr dose-effect (D-E) curve in the cav1 knockout (cav1-/-) mice was shifted to the right compared to wild type controls. In control mice, SR 59230A (β3-blocker) and timolol (β1/β2-blocker) showed similar rightward shifts in the iPr D-E curve that were greater compared to CGP20712A (β1-blocker). In intestine of cav1-/-, SR 59230A and CGP 20712A were without effect; only timolol significantly shifted the D-E curve. BRL 37344 (β3-agonist) was more effective in controls than in cav1-/-intestine, but salbutamol (β2-agonist) relaxed both similarly. Dibutyryl cAMP relaxed intestine in control mice- this was prevented by H-89 (PKA inhibitor). In cav1-/-, H-89 had no effect on relaxation. These results suggest that cav1 knockout reduces β3 (and perhaps β1) receptor function in the small intestine. β2-ARs may partially compensate for this loss. Reduced PKA responses may explain these results. Functional and immunohistochemical experiments are underway to elucidate the interaction between the different β-receptor subtypes and cav1. Supported by CIHR.