title: Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.
abstract: Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize beta(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(2)AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the beta(2)AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of beta(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.
authors.affiliation: Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6.
authors.forename: Dusan
authors.initials: D
authors.lastname: Vobornik
authors.forename: Yanouchka
authors.initials: Y
authors.lastname: Rouleau
authors.forename: Jennifer
authors.initials: J
authors.lastname: Haley
authors.forename: Mahmud
authors.initials: M
authors.lastname: Bani-Yaghoub
authors.forename: Rod
authors.initials: R
authors.lastname: Taylor
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Johnston
authors.forename: John Paul
authors.initials: JP
authors.lastname: Pezacki
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure; D018613:Microscopy, Confocal / Q000379:methods; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria