Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.
G-protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins in the human genome. Events in the GPCR signaling cascade have been well characterized, but the receptor composition and its membrane distribution are still generally unknown. Although there is evidence that some members of the GPCR superfamily exist as constitutive dimers or higher oligomers, interpretation of the results has been disputed, and recent studies indicate that monomeric GPCRs may also be functional. Because there is controversy within the field, to address the issue we have used total internal reflection fluorescence microscopy (TIRFM) in living cells to visualize thousands of individual molecules of a model GPCR, the M(1) muscarinic acetylcholine receptor. By tracking the position of individual receptors over time, their mobility, clustering, and dimerization kinetics could be directly determined with a resolution of approximately 30 ms and approximately 20 nm. In isolated CHO cells, receptors are randomly distributed over the plasma membrane. At any given time, approximately 30% of the receptor molecules exist as dimers, and we found no evidence for higher oligomers. Two-color TIRFM established the dynamic nature of dimer formation with M(1) receptors undergoing interconversion between monomers and dimers on the timescale of seconds.
Division of Physical Biochemistry, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom.
Jonathan A
JA
Hern
Asma H
AH
Baig
Gregory I
GI
Mashanov
Berry
B
Birdsall
John E T
JE
Corrie
Sebastian
S
Lazareno
Justin E
JE
Molloy
Nigel J M
NJ
Birdsall
D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry; D001667:Binding, Competitive; D016466:CHO Cells; D002232:Carbocyanines / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence / Q000379:methods*; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D018727:Muscarinic Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D055503:Protein Multimerization; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D001557:Benzenesulfonates; D002232:Carbocyanines; C495965:Cy3B N-hydroxysuccinimide ester; D005456:Fluorescent Dyes; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; C046338:telenzepine; D010890:Pirenzepine