title: Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.
abstract: The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Latty
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: James H
authors.initials: JH
authors.lastname: Felce
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Laura
authors.initials: L
authors.lastname: Weimann
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Lee
authors.affiliation: Radcliffe Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom. Electronic address: simon.davis@imm.ox.ac.uk.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: dk10012@cam.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D000818:Animals; D016477:Artifacts; D051940:B7-2 Antigen / Q000378:metabolism; D018106:CD28 Antigens / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D004058:Diffusion; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000378:metabolism*; D014162:Transfection
chemical_list: D051940:B7-2 Antigen; D018106:CD28 Antigens; D018343:Receptors, Adrenergic, beta-2; D018080:Receptors, GABA-B