title: Dopamine D2 receptors form higher order oligomers at physiological expression levels.
abstract: G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.
authors.affiliation: Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Guo
authors.forename: Eneko
authors.initials: E
authors.lastname: Urizar
authors.forename: Michaela
authors.initials: M
authors.lastname: Kralikova
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Mobarec
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Marta
authors.initials: M
authors.lastname: Filizola
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008163:Luminescent Measurements; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D003545:Cysteine