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Cell Mol Life Sci ; 78(23): 7557-7568, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34657173

ABSTRACT

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.


Subject(s)
Cell Membrane/metabolism , Receptors, Opioid, delta/chemistry , Receptors, Opioid, delta/metabolism , Receptors, Opioid, mu/chemistry , Receptors, Opioid, mu/metabolism , Single Molecule Imaging/methods , Single-Cell Analysis/methods , Animals , Mice , Protein Conformation , Protein Multimerization , Rats
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