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  • 1
    Keywords: Biochemistry ; Toxicology ; Neurobiology ; Protein Science ; Pharmacology/Toxicology ; Neurobiology ; Springer eBooks
    Description / Table of Contents: Assessment of Conformational State Transitions of Class B GPCRs Using Molecular Dynamics -- Molecular Dynamic Simulations to Probe Water Permeation Pathways of GPCRs -- Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies -- A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties -- Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions -- Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely-Tethered Ligands -- Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assay on GPCRs -- Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-Oligomer Pharmacology -- Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches -- Measuring GPCR Stoichiometry Using Types -1, -2 and -3 Bioluminescence Resonance Energy Transfer-Based Assays -- Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions -- Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters -- Measuring GPCR-Induced Activation of Protein Tyrosine Phosphatases (PTP) Using In-Gel and Colorimetric PTP Assays -- Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay -- Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry -- Monitoring the Aggregation of GPCRs by Fluorescence Microscopy -- Combining RNAi and Immunofluorescence Approaches to Investigate Post-Endocytic Sorting of GPCRs into Multivesicular Bodies -- Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy -- Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy -- Optical Modulation of Metabotropic Glutamate Receptor Type 5 In Vivo Using a Photoactive Drug -- Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice -- Laser Doppler Flowmetry to Study the Regulation of Cerebral Blood Flow by G Protein-Coupled Receptors in Rodents -- Preparation of Pulmonary Artery Myocytes and Rings to Study Vasoactive GPCRs
    Abstract: This detailed volume assembles comprehensive protocols to assist with the study of structural, molecular, cell biological, and in vivo facets of GPCRs, and to enable the development of experimental tools for screening novel GPCR drugs. Sections explore the tweaking of ligands, bioluminescence and FRET approaches, specific GPCR signaling properties, as well as visualization of subcellular compartmentalization. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, G Protein-Coupled Receptor Signaling: Methods and Protocols serves as an ideal reference for life scientists working in a variety of research fields including molecular pharmacology, cell and developmental biology, brain behavior and physiology, drug development and screening. Chapter 4 is available open access under a CC BY 4.0 license via link.springer.com
    Pages: XV, 406 p. 87 illus., 71 illus. in color. : online resource.
    ISBN: 9781493991211
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  • 2
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    New York, NY : Springer
    Keywords: Medicine ; Neurosciences ; Neurochemistry ; Biomedicine ; Neurosciences ; Neurochemistry ; Springer eBooks
    Description / Table of Contents: Genetic and Epigenetic Methods for Analysis of the Dopamine D2 Receptor Gene -- Characterization of D1 Dopamine Receptor Post-Transcriptional Regulation -- Computational Approaches in the Structure-Function Studies of Dopamine Receptors -- Cell-Free Protein Synthesis and Purification of the Dopamine D2 Receptor -- Wnt Ligand Binding to and Regulation of Dopamine D2 Receptors -- Regulation of Pre- and Postsynaptic Protein Phosphorylation by Dopamine D2 Receptors -- Study of Dopamine D1 Receptor Regulation by G Protein-Coupled Receptor Kinases Using Whole-Cell Phosphorylation and Cross-Linking Methods -- Ubiquitination of Dopamine Receptor Studied by Sequential Double Immunoprecipitation -- Dopamine Receptors in the Subthalamic Nucleus: Identification and Localization of D5 Receptors -- MALDI Mass Spectrometry Imaging of Dopamine and PET D1 and D2 Receptor Ligands in Rodent Brain Tissues -- Positron Emission Tomography Imaging of Dopaminergic Receptors in Rats -- Transactivation of Receptor Tyrosine Kinases by Dopamine Receptors -- Dopamine Receptors in Human Embryonic Stem Cell Differentiation -- Calcium and PLC Signaling Through Dopamine Receptors -- Intracellular Trafficking Assays for Dopamine D2-Like Receptors -- Study of Crosstalk Between Dopamine Receptors and Ion Channels -- Study of Dopamine Receptor and Dopamine Transporter Networks in Mice -- Optogenetic Regulation of Dopamine Receptor-Expressing Neurons -- Characterization of D3 Dopamine Receptor Agonist-Dependent Tolerance Property -- Dopamine D1 and D2 Receptors in Chronic Mild Stress: Analysis of Dynamic Receptor Changes in an Animal Model of Depression Using In Situ Hybridization and Autoradiography
    Abstract: To foster a better understanding of dopamine receptor functionality, this detailed volume creates an interface between updated classical methods and new emerging technologies heretofore not available to new or seasoned researchers. Divided in five sections dedicated to experimental approaches investigating different facets of dopaminergic signal transduction, Dopamine Receptor Technologies covers epigenetic and post-transcriptional analysis, computational and biochemical techniques, visualization and imaging methods, molecular and cell biological tools, as well as behavioral assessment. The book, as a part of the popular Neuromethods series, provides insightful step-by-step protocols and methodological reviews that readers will find useful. Practical and versatile, Dopamine Receptor Technologies seeks to aid researchers in developing new pharmacological tools to improve our knowledge of in vivo roles played by each receptor subtype and the synthesis of prospective lead compounds for drug discovery
    Pages: XIV, 379 p. 63 illus., 26 illus. in color. : online resource.
    ISBN: 9781493921966
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The ‘cross-talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP-dependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ɛ, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca2+-independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)-induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild-type (WT) or truncation forms (Δ425, Δ379 and Δ351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA-mediated phosphorylation-dependent desensitization of D1A receptors. However, the longer truncation mutant Δ351 failed to undergo detectable DA-induced phosphorylation while exhibiting DA-induced desensitization features similar to the shorter truncation mutant Δ379. These data potentially suggest a novel role for a receptor phosphorylation-independent process in the DA-promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA-mediated receptor endocytosis. Additionally, time-course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation or kobs, a parameter linked to the receptor-G protein coupling status. In agreement with the desensitization data, Δ425- and Δ379-expressing cells exhibit an increase of kobs in comparison with WT-expressing cells. Nevertheless, Δ351-expressing cells, which harbor similar desensitization features of Δ379-expressing cells, display no change in kobs when compared with WT-expressing cells. Our results suggest that a defective DA-induced endocytosis may hamper Δ351 resensitization and concomitant increase in kobs. Thus, our study showing that specific D1A receptor CT sequences regulate DA-induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Δ425, Δ379, Δ351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys351 and Asp425 may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Δ351 is significantly increased in comparison with WT, Δ425 and Δ379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Δ351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys351 may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Δ351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.
    Type of Medium: Electronic Resource
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