title: Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.
abstract: BACKGROUND
  Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared.
  
  
  RESULTS
  The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods.
  
  
  CONCLUSIONS
  Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.
authors.affiliation: The Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
authors.forename: Eva
authors.initials: E
authors.lastname: Biener
authors.forename: Madia
authors.initials: M
authors.lastname: Charlier
authors.forename: V Krishnan
authors.initials: VK
authors.lastname: Ramanujan
authors.forename: Nathalie
authors.initials: N
authors.lastname: Daniel
authors.forename: Avital
authors.initials: A
authors.lastname: Eisenberg
authors.forename: Christian
authors.initials: C
authors.lastname: Bjørbaek
authors.forename: Brian
authors.initials: B
authors.lastname: Herman
authors.forename: Arieh
authors.initials: A
authors.lastname: Gertler
authors.forename: Jean
authors.initials: J
authors.lastname: Djiane
mesh_terms: D000818:Animals; D001704:Biopolymers / Q000737:chemistry; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D020738:Leptin / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D050796:STAT3 Transcription Factor / Q000378:metabolism
chemical_list: D001704:Biopolymers; C485182:Cyan Fluorescent Protein; C515448:LEPR protein, human; D020738:Leptin; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C515447:leptin receptor, mouse; D049452:Green Fluorescent Proteins; D008156:Luciferases