The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and
controversial. Estimates of size from numerous pharmacological, biochemical and biophysical
studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers
to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present
investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been
examined by two methods. Both are based on the efficiency of Förster resonance energy transfer
(FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral
deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing
eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then
was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for
the transfer of energy between single donors and acceptors within an oligomer of given size (n).
Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging
provided an independent estimate of Ep that was in close agreement with the model-based value
when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2
muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and
the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from
34–40 cells expressing each receptor were compared with those predicted for populations of dimers,
trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined
data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors
were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers
of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist
largely or wholly as constitutive tetramers.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/65486 |
| Date | 19 June 2014 |
| Creators | Pisterzi, Luca Francis |
| Contributors | Wells, James W |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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