Dataset Information

High-Affinity Functional Fluorescent Ligands for Human ?-Adrenoceptors.

ABSTRACT: Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting ?-adrenergic receptor (?AR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates nanomolar binding affinity and up to nine-fold ?2AR selectivity over ?1AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of ?2ARs stained at the native expression level on pancreatic CAPAN cells provides two-fold improvement in lateral optical resolution over confocal mode and reveals the formation of receptor microdomains. These probes retain their functional (agonist or antagonist) properties, allowing simultaneous modulation of cyclic adenosine monophosphate (cAMP) levels and receptor internalization as well as imaging receptor localization.

SUBMITTER: Mitronova GY

PROVIDER: S-EPMC5614969 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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High-Affinity Functional Fluorescent Ligands for Human β-Adrenoceptors.

Mitronova Gyuzel Y GY Lukinavičius Gražvydas G Butkevich Alexey N AN Kohl Tobias T Belov Vladimir N VN Lehnart Stephan E SE Hell Stefan W SW

Scientific reports 20170926 1

Visualization of the G-protein coupled receptor (GPCR) is of great importance for studying its function in a native cell. We have synthesized a series of red-emitting fluorescent probes targeting β-adrenergic receptor (βAR) that are compatible with confocal and Stimulated Emission Depletion (STED) microscopy as well as with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay in living cells. The probe based on the agonist BI-167107 and fluorescent dye KK114 demonstrates ...[more]

PMID: 28951558

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Project description:BackgroundThere are three turkey β-adrenoceptors: the original turkey β-adrenoceptor from erythrocytes (tβtrunc, for which the X-ray crystal structure has recently been determined), tβ3C and tβ4C-receptors. This study examined the similarities and differences between these avian receptors and mammalian receptors with regards to binding characteristics and functional high and low affinity agonist conformations.Methodology/principal findingsStable cell lines were constructed with each of the turkey β-adrenoceptors and 3H-CGP12177 whole cell binding, CRE-SPAP production and (3)H-cAMP accumulation assays performed. It was confirmed that the three turkey β-adrenoceptors are distinct from each other in terms of amino acid sequence and binding characteristics. The greatest similarity of any of the turkey β-adrenoceptors to human β-adrenoceptors is between the turkey β3C-receptor and the human β2-adrenoceptor. There are pharmacologically distinct differences between the binding of ligands for the tβtrunc and tβ4C and the human β-adrenoceptors (e.g. with CGP20712A and ICI118551). The tβtrunc and tβ4C-adrenoceptors appear to exist in at least two different agonist conformations in a similar manner to that seen at both the human and rat β1-adrenoceptor and human β3-adrenoceptors. The tβ3C-receptor, similar to the human β2-adrenoceptor, does not, at least so far, appear to exist in more than one agonist conformation.Conclusions/significanceThere are several similarities, but also several important differences, between the recently crystallised turkey β-adrenoceptor and the human β-adrenoceptors. These findings are important for those the field of drug discovery using the recently structural information from crystallised receptors to aid drug design. Furthermore, comparison of the amino-acid sequence for the turkey and human adrenoceptors may therefore shed more light on the residues involved in the existence of the secondary β-adrenoceptor conformation.

Dataset Information

High-Affinity Functional Fluorescent Ligands for Human ?-Adrenoceptors.

Publications

High-Affinity Functional Fluorescent Ligands for Human β-Adrenoceptors.

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