Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Cell Mol Life Sci. 2021 Dec;78(23):7557-7568. doi: 10.1007/s00018-021-03963-y. Epub 2021 Oct 17.

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.

Keywords: Dimerization affinity; G protein-coupled receptors; Monomer-dimer equilibrium; Opioid receptors; Single-molecule imaging.

MeSH terms

  • Animals
  • Cell Membrane / metabolism*
  • Mice
  • Protein Conformation
  • Protein Multimerization
  • Rats
  • Receptors, Opioid, delta / chemistry*
  • Receptors, Opioid, delta / metabolism*
  • Receptors, Opioid, mu / chemistry*
  • Receptors, Opioid, mu / metabolism*
  • Single Molecule Imaging / methods*
  • Single-Cell Analysis / methods*

Substances

  • Receptors, Opioid, delta
  • Receptors, Opioid, mu