title: TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.
abstract: Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family.
  
  
  CONCLUSIONS
  the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Brejchová
authors.affiliation: J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic v.v.i., Dolejškova 2155/3, 18223 Prague 8, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Sýkora
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Ostašov
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Merta
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Roubalová
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Janáček
authors.affiliation: J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic v.v.i., Dolejškova 2155/3, 18223 Prague 8, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic. Electronic address: svobodap@biomed.cas.cz.
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
mesh_terms: D000465:Algorithms; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D004058:Diffusion; D004161:Diphenylhexatriene / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D036681:Fluorescence Recovery After Photobleaching; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D011485:Protein Binding; D021381:Protein Transport; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018025:Receptors, Thyrotropin-Releasing Hormone; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004161:Diphenylhexatriene; D002784:Cholesterol; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: Cholesterol; DPH fluorescence; FRAP; G protein coupling; RICS; TRH-R-eGFP mobility