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Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family

Abstract

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased “water-accessible space” within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by “antibody feeding” method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

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Abbreviations

AC:

adenylyl cyclase

β-CDX:

β-cyclodextrin

CLSM:

confocal laser scanning microscopy

DADLE:

[D-Ala2, D-Leu5]-enkephalin

DMEM:

Dulbecco’s modified Eagle’s medium

δ-OR:

δ-opioid receptor

δ-OR-Gi1α (C351-I351) cells:

HEK293 cells stably expressing PTX-insensitive δ-OR-Gi1α (C351-I351) fusion protein

DPH:

1,6-diphenyl-1,3,5-hexatriene

FBS:

fetal bovine serum

FLAG-δ-OR:

NH2-terminally FLAG epitope-tagged δ-OR

FLAG-δ-OR cells:

HEK293T cells transiently expressing FLAG-δ-OR

FLIM:

fluorescence lifetime imaging microscopy

GFP:

green fluorescent protein

G protein:

trimeric guanine nucleotide binding protein

GPCR:

G protein-coupled receptor

HEK293 cells:

human embryonic kidney cells

κ-OR:

κ-opioid receptor

methyl-β-CDX:

methyl-β-cyclodextrin

MD:

membrane domains

μ-OR:

μ-opioid receptor

Myc-OTR:

NH2-terminally Myc epitope-tagged OTR

Myc-OTR cells:

HEK293T cells transiently expressing Myc-OTR

Na, K-ATPase:

sodium plus potassium-activated, ouabain-sensitive adenosine triphosphatase (EC 3.6.3.9)

NCS:

newborn calf serum

OTR:

oxytocin receptor

PBS:

phosphate-buffered saline

PM:

plasma (cell) membrane

PMSF:

phenylmethylsulfonyl fluoride

PNS:

post-nuclear supernatant

PTX:

pertussis toxin

TMA-DPH:

(1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate).

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Acknowledgments

This work was supported by Czech Science Foundation (P207/12/0919) and with institutional support RVO: 67985823. The stay of Jana Brejchova in the laboratory of Prof. Marco Parenti (University of Milano-Bicocca, Monza, Italy) was supported by PROGRAM EXTRAPLUS financed by Fondazione Cariplo. The authors thank to Francesca Guzzi for the preparation of the cell line stably expressing FLAG-δ-OR, valuable advices, and kind help in the course of J.B. stay in Monza.

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Supplementary Fig. 1
figure10

The effect of cholesterol depletion on agonist-induced internalization of FLAG-δ-OR - supporting micrographs for the results depicted in Fig. 1a. In order to label cell surface pool of FLAG-δ-OR, HEK293T cells transiently expressing FLAG-δ-OR were pre-incubated with monoclonal antibodies against the extracellular FLAG tag under non-permeable conditions as described in Methods. Subsequently, cells were exposed to serum-free DMEM (control), 10 mM β-CDX in serum-free DMEM (β-CDX), 100 nM DADLE in serum-free DMEM (DADLE), or 10 mM β-CDX in serum-free DMEM containing 100 nM DADLE (DADLE + β-CDX) for 30 min and fixed. After fixation, cells were stained with Alexa Fluor 488-conjugated secondary antibodies under permeable conditions and imaged with confocal laser scanning microscopy. Groups of 4 micrographs per each experimental condition are displayed. The arrangement of the groups with respect to the experimental conditions is the same as in Fig. 1a, i.e., group of micrographs in the upper left part of the figure represent (control), group in the upper right part represent (β-CDX), group in the lower left part represent (DADLE), and group in the lower right part represent (DADLE + β-CDX). (GIF 136 kb)

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Brejchova, J., Vosahlikova, M., Roubalova, L. et al. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family. J Bioenerg Biomembr 48, 375–396 (2016). https://doi.org/10.1007/s10863-016-9667-7

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Keywords

  • Cholesterol
  • Plasma membrane
  • δ-opioid receptor
  • Internalization
  • Rab proteins