Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells.
Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.
Huang
Peng
P
Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, 3420 N Broad Street, Philadelphia, PA 19140, United States.
Xu
Wei
W
Yoon
Su-In
SI
Chen
Chongguang
C
Chong
Parkson Lee-Gau
PL
Liu-Chen
Lee-Yuan
LY
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D051242:Caveolin 1 / Q000378:metabolism; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004174:Diprenorphine / Q000494:pharmacology; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000494:pharmacology; D005048:Etorphine / Q000494:pharmacology; D005677:G(M1) Ganglioside / Q000378:metabolism; D006822:Hybrid Cells; D007981:Levorphanol / Q000494:pharmacology; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009270:Naloxone / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D000924:Anticholesteremic Agents; D051242:Caveolin 1; D008565:Membrane Proteins; D009842:Oligopeptides; D017465:Receptors, Opioid, delta; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; C060209:deltorphin II, Ala(2)-; D004174:Diprenorphine; D007981:Levorphanol; D009270:Naloxone; D005677:G(M1) Ganglioside; D005048:Etorphine; D020881:Enkephalin, D-Penicillamine (2,5)-; D002784:Cholesterol