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 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 nanomolar binding affinity and up to nine-fold β2AR selectivity over β1AR. Carazolol-derived probes are fluorogenic and allow no-wash imaging experiments. STED microscopy of β2ARs 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.
Highlights
Yes for the high quality staining of β-adrenergic receptor (βAR) even with bulky functional groups appended at the position 3 of BI-167107 (Fig. 1e, Table 1).We compared the efficiencies of carazolol-KK114 (24) and of commercial (S)-carazolol fluorescent derivative ab118171 (Abcam plc) as cyclic adenosine monophosphate (cAMP) production inhibitors in HEK293 cells expressing the fluorescent biosensor TEpacVV (Supplementary Fig. 4). ab118171 is a red-emitting β1AR and β2AR antagonist bearing a BODIPY 630/650 fluorophore (Fig. 1e)
Our goal was to synthesize fluorescent βAR probes that are applicable in optical microscopy and we focused on the preparation of BI-167107 derivatives with various linkers and fluorescent ligands attached to the position 3 of the phenyl ring
We have designed, synthesized and characterized five red-emitting fluorescent ligands acting as functional agonists and antagonists of the human βAR receptors
Summary
Ab118171 is a red-emitting (λabs = 633 nm, λem = 650 nm) β1AR and β2AR antagonist bearing a BODIPY 630/650 fluorophore (Fig. 1e). Both fluorescent probes were able to reduce cAMP concentration in isoprenaline-stimulated cells as judged from decrease of TEpacVV sensor CFP/FRET ratio (Supplementary Fig. 4). If the measurements were performed 30 min after addition of the ligands, the Kiapp of carazolol-KK114 (24) decreased to 29.3 ± 15.0 nM (Fig. 3b) This change might be due to the complex processes involving receptor desensitization, internalization and/or activation of cAMP phosphodiesterases[34], and similar phenomena were observed earlier[38]
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