Ste2p Under the Microscope: the Investigation of Oligomeric States of a Yeast G Protein-Coupled Receptor
| Autor: | Cagdas D Son, Sara Mingu-Akmete, Orkun Cevheroğlu, Merve Murat |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Chemistry Saccharomyces cerevisiae Ligand (biochemistry) Receptors G-Protein-Coupled Surfaces Coatings and Films Green fluorescent protein Bimolecular fluorescence complementation Förster resonance energy transfer Mating of yeast Receptors Mating Factor Fluorescence Resonance Energy Transfer Materials Chemistry Biophysics Physical and Theoretical Chemistry mCherry Receptor Protein Binding G protein-coupled receptor |
| Zdroj: | The Journal of Physical Chemistry B. 125:9526-9536 |
| ISSN: | 1520-5207 1520-6106 |
| DOI: | 10.1021/acs.jpcb.1c05872 |
| Popis: | Oligomerization of G protein-coupled receptors (GPCRs) may play important roles in maturation, internalization, signaling, and pharmacology of these receptors. However, the nature and extent of their oligomerization is still under debate. In our study, Ste2p, a yeast mating pheromone GPCR, was tagged with enhanced green fluorescent protein (EGFP), mCherry, and with split florescent protein fragments at the receptor C-terminus. The Forster resonance energy transfer (FRET) technique was used to detect receptors' oligomerization by calculating the energy transfer from EGFP to mCherry. Stimulation of Ste2p oligomers with the receptor ligand did not result in any significant change on observed FRET values. The bimolecular fluorescence complementation (BiFC) assay was combined with FRET to further investigate the tetrameric complexes of Ste2p. Our results suggest that in its quiescent (nonligand-activated) state, Ste2p is found at least as a tetrameric complex on the plasma membrane. Intriguingly, receptor tetramers in their active form showed a significant increase in FRET. This study provides a direct in vivo visualization of Ste2p tetramers and the pheromone effect on the extent of the receptor oligomerization. |
| Databáze: | OpenAIRE |
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