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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


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


N-glycosylation is a common post-translational modification of G protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β2 adrenergic receptor (β2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus and Asn187 at the second extracellular loop (ECL2). Here we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G protein dependent signaling, β-arrestin2 recruitment and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β2 AR dimerization and its effects on receptor signaling was similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization and function of GPCRs. This article is protected by copyright. All rights reserved.

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


G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.

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


Mu opioid receptor (MOR) agonists are often used to treat severe pain, but can result in adverse side effects. To circumvent systemic side effects, targeting peripheral opioid receptors is an attractive alternative treatment for severe pain. Activation of the delta opioid receptor (DOR) produces similar analgesia with reduced side effects. However, until primed by inflammation, peripheral DOR is analgesically incompetent, raising interest in the mechanism. We recently identified a novel role for G protein-coupled receptor kinase 2 (GRK2) that renders DOR analgesically incompetent at the plasma membrane. However, the mechanism that maintains constitutive GRK2 association with DOR is unknown. Protein kinase A (PKA) phosphorylation of GRK2 at Ser685 targets it to the plasma membrane. A-kinase anchoring protein 79/150 (AKAP), residing at the plasma membrane in neurons, scaffolds PKA to target proteins to mediate downstream signal. Therefore, we sought to determine whether GRK2-mediated DOR desensitization is directed by PKA via AKAP scaffolding. Membrane fractions from cultured rat sensory neurons following AKAP siRNA-transfection and from AKAP-knockout mice, had less PKA activity, GRK2 Ser685 phosphorylation, and GRK2 plasma membrane targeting than controls. Site-directed mutagenesis revealed that GRK2 Ser685 phosphorylation drives GRK2s association with plasma membrane-associated DOR. Moreover, overexpression studies with AKAP mutants indicated that impaired AKAP-mediated PKA scaffolding significantly reduces DOR-GRK2 association at the plasma membrane and consequently increases DOR activity in sensory neurons without a priming event. These findings suggest that AKAP scaffolds PKA to increase plasma membrane targeting and phosphorylation of GRK2 to maintain DOR analgesic incompetence in peripheral sensory neurons.

Concepts: Protein, Signal transduction, Receptor, Opioid, Opioid receptor, G protein-coupled receptor, Buprenorphine, Beta-2 adrenergic receptor


G protein-coupled receptors (GPCRs) are important modulators of many physiological functions and excellent drug targets for many diseases. However, to study the functions of endogenous GPCRs is still a challenging task, partially due to the low expression level of GPCRs and the lack of highly potent and selective GPCR antibodies. Overexpression or knock-in of tagged GPCRs, or knockout of specific GPCRs in mice, are common strategies used to study the in vivo functions of these receptors. However, generating separate mice carrying tagged GPCRs or conditional alleles for GPCRs is labor intensive, and requires additional breeding costs. Here we report the generation of mice carrying an HA-tagged DOR (delta opioid receptor) flanked by LoxP sequences at the endogenous DOR locus using a single recombination step, aided by the TALEN system. These animals can be used directly to study the expression, localization, protein-protein interaction and signal transduction of endogenous DOR using anti-HA antibodies. By crossing with mice expressing tissue-specific Cre, these mice can also generate offspring with DOR knockout within specific tissues. These mice are powerful tools to study the in vivo functions of DOR. Furthermore, the gene modification strategy could also be used to study the functions of many other GPCRs.

Concepts: Protein, Gene, Signal transduction, Opioid receptor, G protein-coupled receptor, Metabotropic glutamate receptor, G protein, 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