ORCID Profile
0000-0001-8514-0827
Current Organisation
University of Nottingham
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Publisher: American Chemical Society (ACS)
Date: 16-12-2015
DOI: 10.1021/ACS.JMEDCHEM.5B01664
Abstract: Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pKi values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology.
Publisher: Cold Spring Harbor Laboratory
Date: 29-06-2020
DOI: 10.1101/2020.06.29.171512
Abstract: The β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical GPCR for biophysical studies. Solubilisation of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilises membrane proteins. Use of hiphilic copolymers is a promising strategy to solubilise membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β 2 AR in the polymer Diisobutylene maleic acid (DIBMA). We demonstrate that β 2 AR remains functional in the DIBMA lipid particle (DIBMALP) and shows improved thermal stability compared to the n-Dodecyl-β-D-Maltopyranoside (DDM) detergent solubilised β 2 AR. This unique method of extracting β 2 AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilising mutations and the use of detergents, particularly for functional biophysical studies.
Publisher: Wiley
Date: 12-2041
Publisher: Wiley
Date: 02-2010
Publisher: The Company of Biologists
Date: 15-02-2012
DOI: 10.1242/JCS.091090
Abstract: The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 19-01-2010
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 22-06-2010
Publisher: Wiley
Date: 29-06-2011
DOI: 10.1096/FJ.11-186296
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Stephen Briddon.