Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Synergistic anti-tumor effects of lenalidomide and gefitinib by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

Gefitinib is commonly used to be the first-line therapy for advanced non-small cell lung cancer (NSCLC). Therapeutic effect of gefitinib is reduced due to acquired resistance, and combined treatment is recommended. In this research, we planned to explore the impacts of combined treatment of lenalidomide and gefitinib on gefitinib-sensitive or -resistant NSCLC cells. The co-treatment results demonstrated that enhanced antitumor impact on NSCLC cell growth, migration, invasion, cell cycle process and apoptosis. The tumor-bearing mouse models were established using PC9/GR cells. In vivo assays also showed that lenalidomide and gefitinib synergistically inhibited mouse tumor growth along increased the survival of mice. ADRB2 was identified as a lowly expressed gene in PC9/GR cells and LUAD tumor tissues. LUAD patients with high ADRB2 expression were indicated with favorable survival outcomes. Moreover, ADRB2 was upregulated in lenalidomide and/or gefitinib-treated PC9/GR cells. ADRB2 deficiency partially offsets the suppressive impacts of lenalidomide and gefitinib co-treatment on the viability and proliferation of PC9/GR cells. Additionally, lenalidomide and gefitinib cotreatment significantly inactivated the mTOR/PI3K/AKT signaling pathway compared with each treatment alone. Rescue assays were performed to explore whether lenalidomide and gefitinib synergistically inhibited the growth of PC9/GR cells via the PI3K/AKT pathway. PI3K activator SC79 significantly restored reduced cell proliferation, migration and invasion along with elevated cell cycle arrest and apoptosis caused by lenalidomide and gefitinib cotreatment. In conclusion, lenalidomide and gefitinib synergistically suppressed LUAD progression and attenuated gefitinib resistance by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

The association of gene polymorphisms in catechol-O'methyltransferase (COMT) and ß2-adrenergic receptor (ADRB2) with temporomandibular joint disorders.

OBJECTIVE: Temporomandibular disorder (TMD) has a multifactorial etiology that includes environmental, psychological, and genetic factors. This study aimed to evaluate the possible relationship between polymorphisms in Catechol-O-methyltransferase (COMT) and ß2-adrenergic receptor (ADRB2) genes with TMD. DESIGN: This observational case-control study included 80 patients and 70 healthy controls. The diagnosis of TMD was made using the diagnostic criteria for TMD and the following TMD categories were used for the case group: muscular TMD and articular TMD (disc displacement and arthralgia). A genotyping study of gene polymorphisms in COMT (rs 9332377) and ADRB2 (rs20530449) was performed from genomic DNA isolated from blood. The chi-square test was used to analyze the relationships. P < 0.05 was accepted as a significant difference. RESULTS: The polymorphic TT and CT genotype for COMT (rs rs9332377) was significantly higher in the articular TMD group while the non-polymorphic CC genotype was significantly lower in the articular TMD group (P < 0.05). Regarding ADRB2 (rs20530449), the polymorphic GG genotype was similarly considerably more common in the articular TMD group (p < 0.05). In addition, the T allele in the COMT (rs rs9332377) gene was found to be significantly higher in the articular TMD group (p < 0.05). CONCLUSIONS: In the Turkish population, gene polymorphisms in COMT (rs9332377) and ADRB2 (rs2053044) were associated with articular TMD. This study supports the hypothesis that changes in COMT and ADRB2 genes may play a role in temporomandibular joint pain and predisposition to TMD.

Genetic association between ADRB2 rs1042713 and elite athletic performances in the Korean population.

Athletic performance is a multifactorial trait influenced by environmental and genetic factors. Previous studies have identified various genes associated with athletic performance, including the ß2-adrenergic receptor (ADRB2) gene, which has been consistently shown to be linked with elite athletic performance in diverse populations. The ADRB2 gene is known to play a key role in various biological systems, including cardiovascular, pulmonary, metabolic, and musculoskeletal functions. It acts by interacting with adrenaline. In particular, the ADRB2 rs1042713 (A > G) polymorphism has been associated with cardiovascular and respiratory functions. In addition, the association between the ADRB2 rs1042713 polymorphism and athletic performance has been reported. Thus, we conducted a case-control study to analyze the genetic association with ADRB2 rs1042713 polymorphism with 150 elite athletes, 116 college athletes, and 145 controls (control I) in the Korean population. The genotypes were determined by PCR-RFLP. As a result, we found significant differences in the distributions of genotype (p = 0.005) and allele (p = 0.002) frequencies between elite athletes and the control Ⅱ (control I + college athletes). We also found that the ADRB2 rs1042713 G/G genotype [odds ratio (OR) 2.42, 95% CI 1.384-4.235, p = 0.002] and the G allele (OR 1.58, 95% CI 1.184-2.098, p = 0.002) were significantly associated with elite athletic performance. Additionally, we observed a gender-specific association in female elite athletic performance (p = 0.0002 and p = 0.0002, respectively). In conclusion, our results suggest that the ADRB2 rs1042713 polymorphism may be associated with elite athletic performance in the Korean population. To validate these findings, additional studies with larger samples, including elite athletes from various sports types and diverse ethnic origins are needed.

Prediction of asthma using a four-locus gene model including IL13, IL4, FCER1B, and ADRB2 in children of Kazak nationality.

BACKGROUND: To study whether the four locus gene model consisting of ADRB2 rs1042713, IL4 rs2243250, FCER1B rs569108 and L13 rs20541 can predict asthma of the Kazak children in Xinjiang, China. METHODS: Four single nucleotide polymorphisms about the 4 genes were genotyped in asthma group and control group of Han children and Kazak children respectively. The frequencies of different genotypes and alleles were compared between the asthma group and the control group in the two nationalities. Different risk genotypes for asthma were evaluated in the two nationalities. RESULTS: The differences about frequencies of genotypes in ADRB2 rs1042713 and IL4 rs2243250 and IL13 rs20541 between asthma group and control group were statistically significant in Han children, as were the frequencies of alleles in the 3 single nucleotide polymorphisms, but there were no statistical differences in FCER1B rs569108(P > 0.05). For the Kazak children, no differences were existed among all the genotypes and alleles in asthma group and control group. For the Han children, more children were asthma high risk genotype in the asthma group than those in the control group and no difference was found in the Kazak children. CONCLUSIONS: The four locus gene model consisting of ADRB2 rs1042713, IL4 rs2243250, FCER1B rs569108 and L13 rs20541 can predict asthma of Han children but not for the Kazak children in Xinjiang, which illustrating that the difference of asthma prevalence between different races is closely related to the genetic background.

Evaluation value and mechanism of ADRB2 and FCER1B gene polymorphisms in preterm infants with congenital respiratory diseases.

In this study, we observed the value of ADRB2 and FCER1B gene polymorphisms in evaluating congenital respiratory diseases in preterm infants (PTIs), analyzed their effects on airway smooth muscle cells (ASMCs), and preliminarily discussed the underlying mechanism. First, we placed 64 healthy PTIs (control group) and 45 PTIs with congenital respiratory diseases (research group) born at our hospital from April 2021 to June 2023 were selected as the research subjects. Through testing, we found that the carriers of AA genotype of the polymorphic marker rs1042713 of the ADRB2 gene and that of the rs569108 locus of the FCER1B gene were less in the research group compared with the control group (P<0.05). Preterm infants carrying the GG genotype had a 2.887-fold (P<0.05) increased risk of developing congenital respiratory disease under the recessive model at the rs1042713 locus of the ADRB2 gene. Under the dominant model, preterm infants who did not carry the AA genotype had a 3.070-fold (P<0.05) increased risk of developing congenital respiratory disease. Subsequently, the constructed abnormal expression vectors of ADRB2 and FCER1B were transfected into ASMCs to examine changes in cell activity and pyroptosis. We found that up-regulating ADRB2 and FCERIB expression promoted ASMC proliferation and inflammatory reactions, inhibited apoptosis, and accelerated pyroptosis (P<0.05); silencing their expression, however, led to the opposite effect. In conclusion, the ADRB2 and FCERIB gene polymorphisms are strongly correlated with congenital respiratory diseases, which can provide a reference for clinical evaluation of congenital respiratory diseases in PTIs.

Molecular determinants of ligand efficacy and potency in GPCR signaling.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) bind to extracellular ligands and drugs and modulate intracellular responses through conformational changes. Despite their importance as drug targets, the molecular origins of pharmacological properties such as efficacy (maximum signaling response) and potency (the ligand concentration at half-maximal response) remain poorly understood for any ligand-receptor-signaling system. We used the prototypical adrenaline-ß2 adrenergic receptor-G protein system to reveal how specific receptor residues decode and translate the information encoded in a ligand to mediate a signaling response. We present a data science framework to integrate pharmacological and structural data to uncover structural changes and allosteric networks relevant for ligand pharmacology. These methods can be tailored to study any ligand-receptor-signaling system, and the principles open possibilities for designing orthosteric and allosteric compounds with defined signaling properties.

Genetic polymorphisms in ADRB1, ADRB2 and CYP2D6 genes and response to beta-blockers in patients with acute coronary syndrome.

Betablockers (BBs) are prescribed for ischaemia in patients with acute coronary syndrome (ACS). In Spain, bisoprolol and carvedilol are the most prescribed BBs, but patients often had to discontinue them due to adverse effects. Single nucleotide polymorphisms (SNPs) in ADRB1, ADRB2 and CYP2D6 genes have strong evidence of pharmacogenetic association with BBs in heart failure or hypertension, but the evidence in ACS is limited. Therefore, our study focuses on investigating how these genes influence the response to BBs in ACS patients. We analysed the association between SNPs in ADRB1 Gly389Arg (rs1801253) and Ser49Gly (rs1801252), ADRB2 Gly16Arg (rs1042713) and Glu27Gln (rs1042714), and CYP2D* 6 (*2- rs1080985, *4- rs3892097, *10 - rs1065852) and the occurrence of bradycardia/hypotension events during one year of follow-up. We performed an observational study and included 285 ACS-PCI-stent patients. A first analysis including patients treated with bisoprolol and a second analysis including patients treated with other BBs were performed. We found that the presence of the G allele (Glu) of the ADRB2 gene (rs1042714; Glu27Gln) conferred a protective effect against hypotension-induced by BBs; OR (CI 95%) = 0,14 (0,03-0,60), p < 0.01. The ADRB2 (rs1042713; Gly16Arg) GG genotype could also prevent hypotensive events; OR (CI 95%) = 0.49 (0.28-0.88), p = 0015. SNPs in ADRB1 and CYP2D6 * 2, CYP2D6 * 4 weren´t associated with primary events. The effect of CYP2D6 * 10 does not seem to be relevant for the response to BBs. According to our findings, SNPs in ADRB2 (rs1042713, rs1042714) could potentially affect the response and tolerance to BBs in ACS-patients. Further studies are necessary to clarify the impact of ADRB2 polymorphisms.

Microtubule-Mediated Regulation of ß2AR Translation and Function in Failing Hearts.

BACKGROUND: ß1AR (beta-1 adrenergic receptor) and ß2AR (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 ß-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 ß-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. METHODS: The localization pattern of ß-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on ß-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 ß-AR translation sites in cardiomyocytes. The mechanism by which ß-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. RESULTS: ß1AR and ß2AR mRNAs show differential localization in cardiomyocytes, with ß1AR found in the perinuclear region and ß2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of ß2AR transcripts toward the perinuclear region. The close proximity between ß2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of ß2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both ß1AR and ß2AR 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 ß-AR mRNA redistribution and impaired ß2AR function in failing hearts. CONCLUSIONS: Asymmetrical microtubule-dependent trafficking dictates differential ß1AR and ß2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 ß-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted ß2AR-mediated cyclic adenosine monophosphate signaling.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Regulation of T Helper Cell Type 2 Immune Response by Controlling Beta-2 Adrenergic Receptor in Dendritic Cells of Patients with Allergic Rhinitis.

INTRODUCTION: Allergic diseases are mediated by T helper cell type 2 (Th2) cells, which are differentiated by dendritic cells (DCs). Recently, it was reported that cAMP concentration in DCs is important for inducing allergic responses. However, the regulatory function of cAMP in DCs in Th2 immune responses is unclear. It was hypothesized that the regulation of G protein-coupled receptors (GPCRs) to increase cAMP levels in DCs would reduce Th2 immune responses. METHODS: Human DCs from patients with allergic rhinitis (AR) and from healthy controls were subjected to next-generation sequencing (NGS) to identify potential GPCR. To investigate the functions of GPCR agonists, the in vitro co-culture experiment that THP-1 cells were differentiated into DCs and cultured with human CD4+ T-cells and an AR animal in vivo model were used. RESULTS: Among the GPCRs, the beta-2 adrenergic receptor (ADRB2) of allergic DCs was significantly increased by NGS analysis. The expression of ADRB2 was also increased in Der p 1-treated DCs, which was reduced by treatment with the ADRB2 agonist salbutamol. Salbutamol treatment induced cAMP production in THP-1 derived DCs. In an in vitro co-culture experiment, salbutamol-treated DCs reduced the secretion of Th2 cytokine. In an in vivo AR animal experiment, salbutamol-administered mice showed reduced allergic behavior and Th2 cytokine expression in the nasal mucosa. CONCLUSIONS: The regulation of ADRB2 with salbutamol alleviated the allergic response in vitro DC-T cell co-culture and in vivo AR animal models, suggesting that ADRB2 is a therapeutic target for AR and that ADRB2 agonists may be a promising medication for AR.

Role of beta-2 adrenergic receptor polymorphism (rs1042714) on body weight and glucose metabolism response to a meal-replacement hypocaloric diet.

OBJECTIVES: The beta-2 adrenergic receptor (ADRB2) is involved in energy balance regulation. The objective of our study was to evaluate the role of the rs1042714 genetic variant of ADRB2 gene on weight loss, body composition, and metabolic changes secondary to partial meal replacement (pMR) hypocaloric diet in women with obesity. METHODS: We conducted an interventional study in 95 premenopausal women with body mass index ≥ 35 kg/m2. The subjects received two intakes per day of a normocaloric hyperproteic formula during 12 wk of a pMR diet. Body weight, body mass index, fat mass, waist circumference, lipid profile, fasting insulin levels, and homeostasis model assessment for insulin resistance were determined. All patients were genotyped rs1042714 and evaluated in a dominant model (CC versus CG + GG). RESULTS: Genotype frequencies were 31 (37.3%), 38 (45.8%), and 14 (16.9%) for the CC, CG, and GG genotypes, respectively. We found significant interaction effects between ADRB2 variant and pMR-induced changes (CC versus CG + GG) on body weight (-7.1 ± 0.3 versus -13.5 ± 0.5 kg; P = 0.03), body mass index (-0.9 ± 0.1 versus -1.2 ± 0.2 kg/m2; P = 0.03), fat mass (-4.9 ± 0.5 versus -10.2 ±1.2 kg; P = 0.01), waist circumference (-5.1 ± 0.2 versus -10.1 ± 1.9 cm; P = 0.03), glucose (-5.1 ± 1.3 versus -12.5 ± 2.5 mg/dL; P = 0.03), total cholesterol (-18.1 ± 9.3 versus -33.5 ± 4.5 mg/dL; P = 0.03), low-density lipoprotein cholesterol (-9.1 ± 5.3 versus -24.5 ± 4.1 mg/dL; P = 0.04), triacylglycerol levels (-6.1 ± 5.3 versus -31.5 ± 9.5 mg/dL; P = 0.04), fasting insulin levels (-1.8 ± 0.3 versus -6.3 ± 0.5 IU/L; P = 0.03), and homeostasis model assessment for insulin resistance (-0.6 ± 0.3 versus -1.9 ± 0.5 U; P = 0.03). The odds ratio to improve alteration in glucose metabolism adjusted by age and weight loss throughout the study was 0.26 (95% CI, 0.07-0.95; P = 0.02) in G allele carriers. CONCLUSIONS: The G allele of rs1042714 predicts the magnitude of weight loss resulting from a pMR diet. These adiposity improvements produce a better improvement of insulin resistance and percentage of impaired glucose metabolism in G allele carriers.

Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling.

Intrahepatic cholangiocarcinoma (iCCA) is a highly fatal malignancy with rapidly increasing incidence and mortality worldwide. Currently, gemcitabine-based systemic chemotherapy is the main clinical therapeutic regimen; however, its efficacy is poor, and its mechanism has not been elucidated. In this study, we use a Seahorse Extracellular Flux analyser to measure glycolysis capacity (extracellular acidification rate, ECAR) and oxygen consumption rate (OCR). The glucose uptake or lactic acid content is detected, and the effects of saikosaponin D, an active compound derived from Bupleuri Radix (a traditional Chinese medicine for soothing the liver and relieving depression), on gemcitabine cytotoxicity in norepinephrine-stimulated iCCA cells are analysed. We find that adrenergic signaling plays a fundamental role in chronic stress-induced therapeutic resistance in iCCA. Norepinephrine (NE) and epinephrine (E) enhance the proliferation of iCCA cells and interfere with the response to gemcitabine through activation of the ß2-adrenergic receptor (ADRB2). Furthermore, we find that NE upregulates the expressions of several drug efflux-related genes (such as ABCG2 and MDR1) and promotes glycolysis in iCCA cells. In addition, saikosaponin D reverses the poor response of iCCA cells to gemcitabine by downregulating ADRB2 level. Furthermore, saikosaponin D inhibits drug efflux and glycolysis in iCCA cells by regulating the expressions of MDR1, ABCG2, HK2, and GLUT1. Collectively, saikosaponin D enhances the antitumor effect of gemcitabine by controlling glucose metabolism and drug efflux by inhibiting the ADRB2 signaling. Therefore, the combination of saikosaponin D and gemcitabine may be a potential therapeutic strategy for the treatment of iCCA.

Allosteric modulator potentiates ß2AR agonist-promoted bronchoprotection in asthma models.

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled ß2-adrenergic receptor (ß2AR) agonists (ß2-agonists) promote - with limited efficacy - bronchodilation in asthma. All ß2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a ß2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on ß2AR-mediated bronchoprotection. Consistent with our findings using human ß2ARs, Cmpd-6 allosterically potentiated ß2-agonist binding to guinea pig ß2ARs and downstream signaling of ß2ARs. In contrast, Cmpd-6 had no such effect on murine ß2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated ß2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of ß2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

The impact of genomic variants on patient response to inhaled bronchodilators: a comprehensive update.

INTRODUCTION: The bronchodilator response (BDR) depends on many factors, including genetic ones. Numerous single nucleotide polymorphisms (SNPs) influencing BDR have been identified. However, despite several studies in this field, genetic variations are not currently being utilized to support the use of bronchodilators. AREAS COVERED: In this narrative review, the possible impact of genetic variants on BDR is discussed. EXPERT OPINION: Pharmacogenetic studies of ß2-agonists have mainly focused on ADRB2 gene. Three SNPs, A46G, C79G, and C491T, have functional significance. However, other uncommon variants may contribute to individual variability in salbutamol response. SNPs haplotypes in ADRB2 may have a role. Many variants in gene coding for muscarinic ACh receptor (mAChR) have been reported, particularly in the M2 and, to a lesser degree, M3 mAChRs, but no consistent evidence for a pharmacological relevance of these SNPs has been reported. Moreover, there is a link between SNPs and ethnic and/or age profiles regarding BDR. Nevertheless, replication of pharmacogenetic results is limited and often, BDR is dissociated from what is expected based on SNP identification. Pharmacogenetic studies on bronchodilators must continue. However, they must integrate data derived from a multi-omics approach with epigenetic factors that may modify BDR.

Functional LTCC-ß2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

BACKGROUND: Beta-2 adrenergic receptors (ß2ARs) but not beta-2 adrenergic receptors (ß1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and ß adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. METHODS: Global signaling between LTCCs and ß adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and ß1AR or ß2AR in different membrane microdomains in control and failing cardiomyocytes. RESULTS: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when ß2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and ß2AR was lost. Interestingly, local stimulation of ß1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via ß1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the ß2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream ß1AR and results in an increase in LTCC current. CONCLUSIONS: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via ß2AR, but not ß1AR. This may explain how ß2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

Cost-effectiveness of Arg16Gly in ADRB2 pharmacogenomic-guided treatment for pediatric asthma.

OBJECTIVES: To assess the cost-effectiveness of Arg16Gly ADRB2 pharmacogenomic testing compared with no Arg16Gly ADRB2 testing to guide the use of long-acting ß2 receptor agonist (LABA) in asthma patients aged 1 to 5 years in China. METHODS: This economic evaluation developed a Markov model with four health states (no exacerbation, mild exacerbation, moderate-to-severe exacerbation, and death). Transition probabilities were estimated from the rate of exacerbations, the case-fatality rate of patients hospitalized for exacerbations, and natural mortality. Costs included drug costs and exacerbation management costs. Cost inputs and utilities for each health state were gained from public databases and the literatures. Costs and quality-adjusted life years (QALYs) were estimated for ten years. Deterministic and probabilistic sensitivity analyses were performed. RESULTS: In the base case analysis, in contrast to the group without the genotype test, the incremental total cost was -¥334.7, and the incremental QALY was 0.001 in the Arg16Gly ADRB2 genotyping group. Therefore, the Arg16Gly ADRB2 test group was the dominant strategy for children with asthma in China. The sensitivity analyses showed that the model was relatively stable. CONCLUSION: Arg16Gly ADRB2 testing before using LABA is a cost-effective approach compared with no gene testing for pediatric asthma.