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

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.

Channelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy

“…The estimated dissociation constant for ChR2 C34A/C36A dimers is similar to those previously reported for other transiently dimerizing seven transmembrane helix proteins, class A G‐protein coupled receptors (GPCRs) [68–73] . In GPCRs, dimerization is tightly coupled to the functioning of receptors.…”

“…The estimated dissociation constant for ChR2 C34A/C36A dimers is similar to those previously reported for other transiently dimerizing seven transmembrane helix proteins, class A G-protein coupled receptors (GPCRs). [68][69][70][71][72][73] In GPCRs, dimerization is tightly coupled to the functioning of receptors. Dimerization impacts the trafficking of receptors to the plasma membrane, regulates receptor internalization, [74] affects ligand binding and downstream signaling.…”

Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane

“…Receptor homodimerization has key roles for receptor functionality [28][29][30]. We computed counts per particle (CPP) of KOP-eGFP for analysis of its homodimerization.…”

Opioid Ligands Addressing Unconventional Binding Sites and More Than One Opioid Receptor Subtype

“…Many different methods have been employed to investigate and address opioid receptor dimerization: synthesis of bivalent ligands, development of dimer-selective ligands, fluorescent microscopy techniques, coimmunoprecipitation, development of dimer-selective antibodies etc. [16][17][18][19][20][21][22][23] These studies have demonstrated that the opioid receptors have the capacity to oligomerize between themselves or with non-opioid receptors on the cell membrane. However, whether the ORs are prone to spontaneously organize as dimers in physiological concentrations in native cells, as well as the functional consequences of OR oligomerization in vivo, remain unexplored and controversial due to the complexity of opioid pharmacology.…”

“…Hence, the organization of a GPCR as an oligomer suggests the existence of a new functional unit with a distinct pharmacological profile. Many different methods have been employed to investigate and address opioid receptor dimerization: synthesis of bivalent ligands, development of dimer‐selective ligands, fluorescent microscopy techniques, co‐immunoprecipitation, development of dimer‐selective antibodies etc [16–23] . These studies have demonstrated that the opioid receptors have the capacity to oligomerize between themselves or with non‐opioid receptors on the cell membrane.…”