title: Dopamine receptor oligomerization visualized in living cells.
abstract: G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.
authors.affiliation: Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. brian.odowd@utoronto.ca
authors.forename: Brian F
authors.initials: BF
authors.lastname: O'Dowd
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Ji
authors.forename: Mohammad
authors.initials: M
authors.lastname: Alijaniaram
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Rajaram
authors.forename: Michael M C
authors.initials: MM
authors.lastname: Kong
authors.forename: Asim
authors.initials: A
authors.lastname: Rashid
authors.forename: Tuan
authors.initials: T
authors.lastname: Nguyen
authors.forename: Susan R
authors.initials: SR
authors.lastname: George
mesh_terms: D002069:Butaclamol / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism; D019281:Dimerization; D004298:Dopamine / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D019913:Nuclear Localization Signals; D011487:Protein Conformation; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D018492:Dopamine Antagonists; D019913:Nuclear Localization Signals; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002069:Butaclamol; D004298:Dopamine