| Title: | Oligomerization of yeast alpha-factor receptor detected by fluorescent energy transfer between ligands |
| Author(s): | Connelly SM; Sridharan R; Naider F; Dumont ME; |
| Address: | "Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York. Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York; PhD Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, New York. Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York. Electronic address: mark_dumont@urmc.rochester.edu" |
| DOI: | 10.1016/j.bpj.2021.10.005 |
| ISSN/ISBN: | 1542-0086 (Electronic) 0006-3495 (Print) 0006-3495 (Linking) |
| Abstract: | "G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast alpha-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist alpha-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of alpha-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native alpha-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane" |
| Keywords: | "Fluorescence Resonance Energy Transfer Ligands *Receptors, G-Protein-Coupled Receptors, Mating Factor/genetics *Saccharomyces cerevisiae;" |
| Notes: | "MedlineConnelly, Sara M Sridharan, Rajashri Naider, Fred Dumont, Mark E eng R01 GM022087/GM/NIGMS NIH HHS/ R01 GM059357/GM/NIGMS NIH HHS/ R01 GM084083/GM/NIGMS NIH HHS/ R01 GM114974/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2021/10/11 Biophys J. 2021 Nov 16; 120(22):5090-5106. doi: 10.1016/j.bpj.2021.10.005. Epub 2021 Oct 8" |
