title: Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion.
abstract: Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Brejchová
authors.forename: Jan
authors.initials: J
authors.lastname: Sýkora
authors.forename: Kateřina
authors.initials: K
authors.lastname: Dlouhá
authors.forename: Lenka
authors.initials: L
authors.lastname: Roubalová
authors.forename: Pavel
authors.initials: P
authors.lastname: Ostašov
authors.forename: Miroslava
authors.initials: M
authors.lastname: Vošahlíková
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
mesh_terms: D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D002784:Cholesterol; D019204:GTP-Binding Proteins