MRC National Institute for Medical Research, London
Science for Health
Formation and dissociation of receptor dimers, seen at a single molecule level
16 February 2010
G-protein-coupled receptors (GPCRs) are the largest family of trans-membrane signaling proteins in the human genome. A very small number of GPCRs have been shown unequivocally to exist as constitutive dimers. The results from studies of other GPCRs have also been interpreted in terms of the presence of dimers, or possibly higher oligomers. These conclusions have been disputed by a small number of groups but the underlying dogma that ‘GPCRs are born, live and die as dimers’ has evolved. However some recent studies have suggested that monomeric GPCRs may also be functional and there is no necessity for the presence of dimers. So, there is controversy within the field.
Nigel Birdsall and Justin Molloy (pictured right) in NIMR's Division of Physical Biochemistry have visualized thousands of individual molecules of a model GPCR, the M1 muscarinic acetylcholine receptor using total internal reflection fluorescence microscopy in living cells By tracking the position of individual receptors on the membrane surface in real time, their mobility, clustering and dimerisation kinetics were determined directly. The molecules move randomly over the surface of these cells and absolutely no evidence of receptor oligomers was observed. The lifetime of the receptor dimers is about 1 second, so the interconversion between monomers and dimers is quite rapid. In these experiments, at any given time, about 20-30% of the receptors are present as dimers.
After working on these receptors for over 35 years, it is wonderful to be able to see them as individual molecules rather than as bulk contributors to the generation of new pharmacological data or as a band on a gel. It has been pleasing to be able to contribute to the resolution of a long-standing controversy in the field
Nigel Birdsall
Montage of 100 images showing the transient formation of a muscarinic receptor dimer
Montage of 100 images showing the transient formation of a muscarinic receptor dimer at the cell membrane of a living mammalian cell. The spots of light seen in the images are produced by individual receptors labeled with Cy3B-telenzepine; two individual receptors come together to form a single spot and then separate again when the dimer dissociates. Live CHO cells were imaged every 30ms at 37oC. Each image is 2.2µm across.
Movie, in real time, of individual M1 muscarinic receptors moving on the cell surface.
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Half of the molecules have been labelled with a dye that fluoresces red and half with a dye that fluoresces green. Dimer formation is seen as the formation of a transient yellow spot. The number of spots decreases during the recording because of bleaching of the dye by the laser.
Original article
The research findings are published in full in:
Hern JA, Baig AH, Mashanov GI, Birdsall B, Corrie JE, Lazareno S, Molloy JE, Birdsall NJ. (2010)
Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.
Proceedings of the National Academy of Sciences of the USA, 107(6):2693-8. Publisher abstract
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