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Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations
- Source :
- J Biol Chem
- Publication Year :
- 2020
- Publisher :
- Elsevier BV, 2020.
-
Abstract
- Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.
- Subjects :
- 0301 basic medicine
Agonist
Rhodopsin
Opsin
Light
Protein Conformation
medicine.drug_class
G protein
Biochemistry
Fluorescence
Receptors, G-Protein-Coupled
03 medical and health sciences
GTP-Binding Proteins
medicine
Arrestin
Animals
Amino Acid Sequence
Receptor
Molecular Biology
Fluorescent Dyes
G protein-coupled receptor
030102 biochemistry & molecular biology
biology
Chemistry
Cell Biology
Protein engineering
030104 developmental biology
biology.protein
Biophysics
Protein Binding
Signal Transduction
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 295
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....2679cdd59335dd06ec9bee3e9f3383fa