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Concept: Beta-2 adrenergic receptor


A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

Concepts: Protein, Signal transduction, Cell membrane, G protein-coupled receptor, Metabotropic glutamate receptor, Guanine nucleotide exchange factor, G protein, Beta-2 adrenergic receptor


G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Concepts: Signal transduction, G protein-coupled receptor, Metabotropic glutamate receptor, Arrestin, Cross-link, G protein, G protein-coupled receptors, Beta-2 adrenergic receptor


Aberrant DNA methylation has been observed in the patients with Alzheimer’s disease (AD), a common neurodegenerative disorder in the elderly. OPRD1 encodes the delta opioid receptor, a member of the opioid family of G-protein-coupled receptors. In the current study, we compare the DNA methylation levels of OPRD1 promoter CpG sites (CpG1, CpG2, and CpG3) between 51 AD cases and 63 controls using the bisulfite pyrosequencing technology. Our results show that significantly higher CpG3 methylation is found in AD cases than controls. Significant associations are found between several biochemical parameters (including HDL-C and ALP) and CpG3 methylation. Subsequent luciferase reporter gene assay shows that DNA fragment containing the three OPRD1 promoter CpGs is able to regulate gene expression. In summary, our results suggest that OPRD1 promoter hypermethylation is associated with the risk of AD.

Concepts: DNA, Gene, Genetics, Gene expression, Molecular biology, DNA methylation, Opioid receptor, Beta-2 adrenergic receptor


Genetic polymorphisms in G-protein beta-3 subunit (GNβ3) and beta-2 adrenergic receptor (ADRB2) are associated with pain and gut hypersensitivity, which can overlap with gastroesophageal reflux disease (GERD).

Concepts: Cohort study, Epidemiology, Asthma, Cough, G protein-coupled receptor, Gastroesophageal reflux disease, Adrenergic receptor, Beta-2 adrenergic receptor


The beta-adrenergic receptors coded by the ADRB1, ADRB2 and ADRB3 genes play important roles in mediating metabolic effects, especially lipolysis, insulin resistance and energy balance. This study investigated the expression levels of these three genes in different tissues of Qinchuan cattle by real-time polymerase chain reaction (RT-PCR). Expressed levels of RNA from the ADRB2 gene were generally much higher than for ADRB1 and ADRB3. ADRB1 and ADRB2 expression levels were highest in subcutaneous fat and lower in muscle, whereas ADRB3 expression was higher in muscle tissue. Eight single nucleotide polymorphisms (SNPs) were discovered in 503 Qinchuan cattle by DNA sequencing, containing three missense mutations (g.1148G>C in ADRB1, g.1293C>T and g.1311T>C in ADRB2), four synonymous mutations (g.1054T>C, g.1122C>T and g.1143G>T in ADRB1 and g.506A>G in ADRB3), as well as one mutation in 3'untranslated region (3'UTR) (g.2799G>A in ADRB3). Interestingly, five of them were located in regions predicted to contain multiple repeats of CG nucleotides (CpG islands). Association analysis showed relationships between most of those SNPs or combined haplotypes and carcass traits of Qinchuan cattle. This study association analysis suggests that polymorphisms in these genes might be useful for selection in beef cattle breeding.

Concepts: DNA, Gene, Gene expression, Polymerase chain reaction, Molecular biology, RNA, Adrenergic receptor, Beta-2 adrenergic receptor


The GRK/β-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and β-arrestins on PKC activation. The roles of GRK2 and β-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D2R, D3R, and β2 adrenergic receptor (β2AR). The subdomains involved in the mutual interactions among GRK2, β-arrestin2, and PKCβII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, β-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and β-arrestin2 mutually inhibited the PKCβII autophosphorylation, a hallmark of PKCβII activation. β-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as β-arrestin2. Furthermore, GRK2 facilitated β-arrestin2 ubiquitination, thus to enhance the inhibitory actions of β-arrestin2 on PKCβII activity. Aforementioned processes were also involved in the GRK2/β-arrestin2-mediated inhibition of the D2R, D3R, and β2AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of β-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.

Concepts: Signal transduction, Cell membrane, Receptor, G protein-coupled receptor, Metabotropic glutamate receptor, G protein, G protein-coupled receptors, Beta-2 adrenergic receptor


Proteinase-Activated Receptor-2 (PAR2 ) is a G protein-coupled receptor activated by serine proteinases. We have shown that PAR2 activation in the airways is involved in the development of allergic inflammation and airway hyperresponsiveness (AHR) in acute murine models. We hypothesize that functional inhibition of PAR2 prevents allergic inflammation, AHR and airway remodeling in chronic allergic airway inflammation models. We developed a 12 week model of cockroach extract (CE)-mediated AHR, airway inflammation and remodeling in BALB/c. These mice exhibit AHR, increased numbers of eosinophils in bronchoalveolar lavage (BAL) and increased collagen content in the lung tissue compared to saline controls. Administration of an anti-PAR2 antibody, SAM-11, after the initial development of airway inflammation significantly inhibited all these parameters. Our data demonstrate that PAR2 signaling plays a key role in CE-induced AHR and airway inflammation/remodeling and that targeting PAR2 activation may be a successful therapeutic strategy for allergic asthma. This article is protected by copyright. All rights reserved.

Concepts: Immune system, Signal transduction, Asthma, Lung, Allergy, Chronic, Copyright, Beta-2 adrenergic receptor


G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs.

Concepts: Signal transduction, Cell membrane, Protein kinase, G protein-coupled receptor, Metabotropic glutamate receptor, G protein, Protein kinase A, Beta-2 adrenergic receptor


Delta opioid receptors (δORs) regulate a number of physiological functions, and agonists for this receptor are being pursued for the treatment of mood disorders, chronic pain, and migraine. A major challenge to the development of these compounds is that, like many G-protein coupled receptors (GPCRs), agonists at the δOR can induce very different signaling and receptor trafficking events. This concept, known as ligand-directed signaling, functional selectivity, or biased agonism, can result in different agonists producing highly distinct behavioral consequences. In this chapter, we highlight the in vitro and in vivo evidence for ligand-directed signaling and trafficking at the δOR. A number of biological implications of agonist-directed signaling at the δOR have been demonstrated. Importantly, ligand-specific effects can impact both acute behavioral effects of delta agonists, as well as the long-term adaptations induced by chronic drug treatment. A better understanding of the specific signaling cascades that regulate these differential behavioral effects would help to guide rational drug design, ultimately resulting in δOR agonists with fewer adverse effects.

Concepts: Pharmacology, Signal transduction, Opioid, Agonist, Opioid receptor, Buprenorphine, Beta-2 adrenergic receptor, Delta Opioid receptor


The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans-Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of Phosphoinositide-3 Kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with Nerve Growth Factor (NGF). Depletion of Class II Phosphoinositide 3 Kinase α (PI3K C2A), but not inhibition of Class I PI3K, blocked δR export to comparable levels and attenuated δR-mediated cAMP inhibition. NGF treatment displaced PI3K C2A from the Golgi, and optogenetic recruitment of the PI3K C2A kinase domain to the TGN induced δR export downstream of NGF. Importantly, PI3K C2A expression promotes export of endogenous δR in primary Trigeminal Ganglion neurons. Together, our results identify PI3K C2A as being required and sufficient for δR export and surface delivery in neuronal cells, and suggest that it could be a key modulator of a novel Golgi export checkpoint that coordinates GPCR delivery to the surface.

Concepts: Nervous system, Neuron, Signal transduction, Cell membrane, Opioid receptor, G protein-coupled receptor, Nerve growth factor, Beta-2 adrenergic receptor