Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor–arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization

Cellular, circuit and transcriptional framework for modulation of itch in the central amygdala

Itch is an unpleasant sensation that elicits robust scratching and aversive experience. However, the identity of the cells and neural circuits that organize this information remains elusive. Here, we show the necessity and sufficiency of chloroquine-activated neurons in the central amygdala (CeA) for both itch sensation and associated aversion. Further, we show that chloroquine-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of chloroquine-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send projections to the ventral periaqueductal gray that are critical in modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice

Dopamine D3 receptor dysfunction prevents anti-nociceptive effects of morphine in the spinal cord

Abstract Dopamine (DA) modulates spinal reflexes, including nociceptive reflexes, in part via the D3 receptor subtype. We have previously shown that mice lacking the functional D3 receptor (D3KO) exhibit decreased paw withdrawal latencies from painful thermal stimuli. Altering the DA system in the CNS, including D1 and D3 receptor systems, reduces the ability of opioids to provide analgesia. Here, we tested if the increased pain sensitivity in D3KO might result from a modified μ-opioid receptor (MOR) function at the spinal cord level. As D1 and D3 receptor subtypes have competing cellular effects and can form heterodimers, we tested if the changes in MOR function may be mediated in D3KO through the functionally intact D1 receptor system. We assessed thermal paw withdrawal latencies in D3KO and wild type (WT) mice before and after systemic treatment with morphine, determined MOR and phosphorylated MOR (p-MOR) protein expression levels in lumbar spinal cords, and tested the functional effects of DA and MOR receptor agonists in the isolated spinal cord. In vivo, a single morphine administration (2 mg/kg) increased withdrawal latencies in WT but not D3KO, and these differential effects were mimicked in vitro, where morphine modulated spinal reflex amplitudes (SRAs) in WT but not D3KO. Total MOR protein expression levels were similar between WT and D3KO, but the ratio of pMOR/total MOR was higher in D3KO. Blocking D3 receptors in the isolated WT cord precluded morphine's inhibitory effects observed under control conditions. Lastly, we observed an increase in D1 receptor protein expression in the lumbar spinal cord of D3KO. Our data suggest that the D3 receptor modulates the MOR system in the spinal cord, and that a dysfunction of the D3 receptor can induce a morphine-resistant state. We propose that the D3KO mouse may serve as a model to study the onset of morphine resistance at the spinal cord level, the primary processing site of the nociceptive pathway

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

Complementarity of Δ opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the Δ opioid receptor selective ligand JOM-13 (Tyr–c[ D -Cys–Phe– D -Pen]OH) and the parallel, independent development of a structural model of the Δ receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr 1 residue and in the Phe 3 side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained Δ receptor ligands. Our model of the Δ opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other Δ ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the “alternate” allowed conformer, identified from analysis of the ligands but “disfavored” because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other Δ ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ. © 2000 John Wiley & Sons, Inc. Biopoly 51: 426–439, 1999Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34323/1/5_ftp.pd

Ligand-directed labeling of opioid receptors for covalent attachment of fluorophores or small-molecule probes

Summary: This protocol describes endogenous labeling of opioid receptors (ORs) using a ligand-directed reagent, naltrexamine-acylimidazole compounds (NAI-X). NAI acts by guiding and permanently tagging a small-molecule reporter (X)—such as fluorophores or biotin—to ORs. Here we detail syntheses and uses of NAI-X for OR visualization and functional studies. The NAI-X compounds overcome long-standing challenges in mapping and tracking endogenous ORs as the labeling can be done in situ with live tissues or cultured cells.For complete details on the use and execution of this protocol, please refer to Arttamangkul et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

[mu]-Opioid receptors: ligand-dependent activation of potassium conductance, desensitization, and internalization

μ-Opioid receptor (MOR) desensitization and endocytosis have been implicated in tolerance and dependence to opioids. The efficiency of each process is known to be agonist dependent; however, it is not known what determines the relative efficiency of various agonists at either process. In the present study, homologous MOR desensitization in locus ceruleus (LC) neurons and MOR internalization in HEK293 cells were examined using a series of agonists. The results show that the rank order of this series of agonists was different when comparing the magnitude of hyperpolarization and the ability to cause desensitization in LC neurons. Endocytosis of MOR was also examined in HEK293 cells using the same agonists. The relative ability to cause endocytosis in HEK293 cells correlated with the degree of desensitization in LC cells. This strong correlation suggests that the two processes are closely linked. The results also suggest that agonist efficacy is not necessarily a predictor of the ability to cause MOR desensitization or endocytosis. Identification and characterization of the biophysical properties of agonists that favor desensitization and internalization of receptors will lead to a better understanding of opioid signaling.Veronica A. Alvarez, Seksiri Arttamangkul, Vu Dang, Abdallah Salem, Jennifer L. Whistler, Mark von Zastrow, David K. Grandy, and John T. William