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.

BIOSS Centre for Biological Signalling Studies University of Freiburg Freiburg Germany

CIBSS Centre for Integrative Biological Signalling Studies University of Freiburg Freiburg Germany

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Department of Biomathematics Institute of Physiology Czech Academy of Sciences Prague Czech Republic

Faculty of Biology University of Freiburg Freiburg Germany

Institute of Internal Medicine 4 Medical Center of the University of Freiburg Freiburg Germany

Institute of Pharmaceutical Sciences University of Freiburg Freiburg Germany

Prague Czech Republic

Zobrazit více v PubMed

Milligan G, Ward JW, Marsango S. GPCR homo-oligomerization. Curr Opin Cell Biol. 2019;57:40–47. doi: 10.1016/j.ceb.2018.10.007. PubMed DOI PMC

Cvejic S, Devi LA. Dimerization of the delta opioid receptor: implication for a role in receptor internalization. J Biol Chem. 1997;272:26959–26964. doi: 10.1074/jbc.272.43.26959. PubMed DOI

Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature. 1999;399:697–700. doi: 10.1038/21441. PubMed DOI PMC

George SR et al (2000) Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. J Biol Chem 275:26128–26135 PubMed

Ramsay D, Kellett E, McVey M, Rees S, Milligan G. Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochem J. 2002;365:429–440. doi: 10.1042/bj20020251. PubMed DOI PMC

Wang D, Sun X, Bohn LM, Sadée W. Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer. Mol Pharmacol. 2005;67:2173–2184. doi: 10.1124/mol.104.010272. PubMed DOI

Hern J, et al. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc Natl Acad Sci USA. 2010;107:2693–2698. doi: 10.1073/pnas.0907915107. PubMed DOI PMC

Madl J, et al. Resting state Orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells. J Biol Chem. 2010;285:41135–41142. doi: 10.1074/jbc.M110.177881. PubMed DOI PMC

Kasai RS, et al. Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J Cell Biol. 2011;192:463–480. doi: 10.1083/jcb.201009128. PubMed DOI PMC

Calebiro D, et al. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci USA. 2013;110:743–748. doi: 10.1073/pnas.1205798110. PubMed DOI PMC

Gentzsch C, et al. Selective and wash-resistant fluorescent dihydrocodeinone derivatives allow single-molecule imaging of μ-opioid receptor dimerization. Angew Chem Int Ed. 2020;59:5958–5964. doi: 10.1002/anie.201912683. PubMed DOI PMC

Möller J, et al. Single-molecule analysis reveals agonist-specific dimer formation of μ-opioid receptors. Nat Chem Biol. 2020;16:946–954. doi: 10.1038/s41589-020-0566-1. PubMed DOI

Drakopoulos A, et al. Investigation of inactive-state κ opioid receptor homodimerization via single-molecule microscopy using new antagonistic fluorescent probes. J Med Chem. 2020;63:3596–3609. doi: 10.1021/acs.jmedchem.9b02011. PubMed DOI

Asher WB, et al. Single-molecule FRET imaging of GPCR dimers in living cells. Nat Methods. 2021;18:397–405. doi: 10.1038/s41592-021-01081-y. PubMed DOI PMC

Wehr MC, et al. Monitoring regulated protein-protein interactions using split TEV. Nat Methods. 2006;3:985–993. doi: 10.1038/nmeth967. PubMed DOI

Bishayee S et al (1989) Ligand-induced dimerization of the platelet-derived growth factor receptor. Monomer-dimer interconversion occurs independent of receptor phosphorylation. J Biol Chem 264:11699–11705 PubMed

Choi S, et al. Transmembrane domain-induced oligomerization Is crucial for the functions of syndecan-2 and syndecan-4. J Biol Chem. 2005;280:42573–42579. doi: 10.1074/jbc.M509238200. PubMed DOI

Belyy V, et al. PhotoGate microscopy to track single molecules in crowded environments. Nat Commun. 2017;8:13978. doi: 10.1038/ncomms13978. PubMed DOI PMC

Hu CD, Chinenov Y, Kerppola TK. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell. 2002;9:789–798. doi: 10.1016/S1097-2765(02)00496-3. PubMed DOI

Kodama Y, Hu CD. Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives. Biotechniques. 2012;53:285–298. doi: 10.2144/000113943. PubMed DOI

Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol. 2005;23:102–107. doi: 10.1038/nbt1044. PubMed DOI

Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 2002;296:913–916. doi: 10.1126/science.1068539. PubMed DOI

Sbalzarini IF, Koumoutsakos P. Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol. 2005;151:182–195. doi: 10.1016/j.jsb.2005.06.002. PubMed DOI

Beutel O, et al. Two-dimensional trap for ultrasensitive quantification of transient protein interactions. ACS Nano. 2015;9:9783–9791. doi: 10.1021/acsnano.5b02696. PubMed DOI

Sarabipour S, Hristova K. Mechanism of FGF receptor dimerization and activation. Nat Commun. 2016;7:10262. doi: 10.1038/ncomms10262. PubMed DOI PMC

Stoneman MR, et al. A general method to quantify ligand-driven oligomerization from fluorescence-based images. Nat Methods. 2019;16:493–496. doi: 10.1038/s41592-019-0408-9. PubMed DOI PMC

Uhlén M, et al. Tissue-based map of the human proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419. PubMed DOI

Danke C, et al. Adjusting transgene expression levels in lymphocytes with a set of inducible promoters. J Gene Med. 2010;2:501–515. doi: 10.1002/jgm.1461. PubMed DOI