Abstract
Principal FindingsHEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.SummaryLimited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.
Highlights
Membrane domains/caveolae are plasma membrane (PM) compartments enriched in cholesterol and glycosphingolipids, which are resistant to solubilization by non-ionic detergents such as Triton X-100 at 0°C [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]
The high level of total [32P]GTPase activity per fraction and specific [32P] GTPase activity was detected in “floating”, low-density fractions 1–6 (Fig 1). Membranes recovered in these fractions were collectively termed the low-density membranes, LDM
1) This work describes the simple method for preparation of intermediate forms of low-density, detergent-treated plasma membrane fragments (Brij-58-treated LDM) which are distinct from detergent-untreated PM fragments (LDM) exhibiting the same density but, on the other hand, do not represent the typical detergent-resistant membrane domains (DRMs) isolated in 0.5–1% Triton-X100 at 0°C
Summary
Membrane domains/caveolae are plasma membrane (PM) compartments enriched in cholesterol and glycosphingolipids, which are resistant to solubilization by non-ionic detergents such as Triton X-100 at 0°C [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. Besides the resistance to detergent solubilization, the second characteristic feature of these PM structures is the low buoyant density when exposed to the high centrifugation force. They migrate up to the low-density area equivalent in density to 15– 20% w/v sucrose. The cell homogenate or membrane preparations are treated with Triton X-100 for 60 min at 0–4°C and fractionated by flotation in density gradients [7,8,18] These procedures using the high detergent concentrations (0.5–1%), result in a loss of functional coupling between GPCR and their cognate signaling molecules, trimeric G proteins and adenylyl cyclase (AC) [19]. The low amount of protein in final preparation of membrane domains/caveolae is not sufficient for characterization of GPCR by radioligand binding assays using 3H-labelled ligands (40–60 Ci/mmol) and measurements of G protein response to agonist stimulation by high-affinity [32P]GTPase or [35S]GTPγS binding assays
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