Cannabinoid receptors distribution in mouse cortical plasma membrane compartments
Department of Anatomy, Physiology, Pharmacology; College of Medicine, University of Saskatchewan, 105 Wiggins Road, Health Sciences Bldg. Room GD30.5, Saskatoon, SK S7N 5E5 Canada
College of Pharmacy and Nutrition, University of Saskatchewan, 105 Wiggins Road, Health Sciences Bldg. Room 3B36, Saskatoon, SK S7N 5E5 Canada
Department of Pharmacology, College of Medicine, Dalhousie University, Halifax, NS Canada
Abstract
The type 1 and type 2 cannabinoid receptors (CB1 and CB2 receptors) are class A G protein-coupled receptors (GPCRs) that are activated by endogenous lipids called endocannabinoids to modulate neuronal excitability and synaptic transmission in neurons throughout the central nervous system (CNS), and inflammatory processes throughout the body. CB1 receptor is one of the most abundant GPCRs in the CNS and is involved in many physiological and pathophysiological processes, including mood, appetite, and nociception. CB2 receptor is primarily found on immunomodulatory cells of both the CNS and the peripheral immune system. In this study, we isolated lipid raft and non-lipid raft fractions of plasma membrane (PM) from mouse cortical tissue by using cold non-ionic detergent and sucrose gradient centrifugation to study the localization of CB1 receptor and CB2 receptor. Lipid raft and non-lipid raft fractions were confirmed by flotillin-1, caveolin-1 and transferrin receptor as their protein biomarkers. Both CB1 receptor and CB2 receptor were found in non-raft compartments that is inconsistent with previous findings in cultured cell lines. This study demonstrates compartmentalization of both CB1 receptor and CB2 receptor in cortical tissue and warrants further investigation of CB1 receptor and CB2 receptor compartmental distribution in various brain regions and cell types.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13041-021-00801-x.
Untitled section
Keywords: Type 1 cannabinoid receptor, Type 2 cannabinoid receptor, Lipid rafts
Article notes
Untitled section
Received 2021 Mar 14; Accepted 2021 Jun 2; Collection date 2021.
Introduction
Cannabinoid (CB) receptors are G protein-coupled receptors (GPCRs) that are highly expressed in almost all mammalian tissues. CB receptors are activated by the phytocannabinoid ∆9-tetrahydrocannabinol (Δ9-THC) and endogenous cannabinoids anandamide and 2-arachidonoylglycerol [1]. There are currently two widely-accepted CB receptors: the type 1 cannabinoid receptor (CB1 receptor) and the type 2 cannabinoid receptor (CB2 receptor). CB1 receptor is present in the central nervous system (CNS) and especially in the hippocampus, neocortex, basal ganglia and cerebellum [2, 3]. Interestingly, CB1 receptors are highly enriched in presynaptic and axonal compartments, with their more relevant activity in synaptic sites [4]. The CB1 receptor is involved in cognition, motor function, memory and nociception in the CNS [5]. The CB2 receptor is expressed at highest levels on immunomodulatory cells of the CNS—such as glia—and periphery—such as leukocytes [6, 7]. The expression of CB2 receptor is tightly regulated and induced by inflammatory signals in the microglia and brain-resident macrophages [8, 9].
The CB1 receptor is being intensively studied as a target of interest in a range of CNS disorders [10, 11] including anxiety [12], pain [13, 14], obesity [15, 16], multiple sclerosis [17, 18], nicotine addiction [19, 20], Parkinson disease [21, 22], Alzheimer disease [23] and Huntington disease [24]. The CB2 receptor has been associated with peripheral inflammatory disorders, including nephrotoxicity [25, 26]. Activation of the CB1 receptor at the cell surface typically results in the inhibition of cyclic adenosine monophosphate (cAMP) and the influx of Ca2+ via coupling with Gai/o proteins [27]. Intracellularly-localized CB1 receptors form a subpopulation with different functionalities from their cell surface-localized counterparts [28]. CB1 receptors in the endo/lysosome increase intracellular Ca2+ concentrations by releasing internal stores of Ca2+ and increase lysosomal permeability [29]. Mitochondrial CB1 receptors impair mitochondrial cellular respiration and production of cAMP, thus controlling cellular energy metabolism [28, 30]. Neuronal CB2 receptor has been mainly described as being localized within the postsynaptic region of the hippocampus [31]. Furthermore, previous studies have shown the intracellular localization of CB2 receptor in prefrontal cortical pyramidal neurons where it mediates neuronal excitability by controlling the Ca2+ activated Cl− channels [32]. The CB2 receptor is also localized at the endo/lysosomes and modulates Ca2+ signaling [33].
Lipid rafts are dynamic microdomains in the cell membrane, composed of cholesterol and sphingolipids in comparison with the phospholipid-rich surrounding membranes. These compartments are involved in different cellular functions, including intracellular signaling, cellular polarity, membrane transport and molecule sorting [34–37]. Flotillins and caveolins are two structural proteins of lipid rafts. Acylated proteins and glycosylphosphatidylinositol (GPI)-anchor proteins are preferentially localized in lipid rafts. Cellular prion protein (PrPc) a GPI-anchor protein is specifically resided in lipid raft. Caveolin-1 is predominantly expressed in immature cortical neurons and has association with CB1 receptor [38]. Lipid raft proteins are relatively co-purified with sphingolipids, and cholesterol-rich membranes that are insoluble in cold non-ionic detergents followed by sucrose or Optiprep™ gradient fractionation [39–41]. To better understand the endocannabinoid system, the localization and distribution of CB receptors in the plasma membrane (PM) is important. The distribution of CB receptors in PM of rodent cortical tissue has not been adequately studied. In the present study, we thus investigated localization of CB1 and CB2 receptors in PM compartments of mouse cortices using cold Triton X-100 and sucrose gradient centrifugation method. Protein and lipid contents of PM contribute to distribution pattern of CB receptors in cell membrane. Since protein and lipid contents vary between cell lines and live tissue, we hypothesize that the distribution of CB receptors will be different from those previously reported cell lines studies. We found that the CB1 and CB2 receptors are localized in the non-lipid raft compartment of the PM of mouse cortical tissue.
Materials and methods
Chemicals and antibodies
Antibodies used in these experiments were as follows: rabbit polyclonal anti-CB1 receptor antibody directed against the first 99 amino acids of the receptor (Sigma-Aldrich, IgG, Cat# C1108, lot# SLCD8394) (dilution of 1:250) [42]; rabbit polyclonal anti-CB2 receptor antibody directed against the first 32 amino acids of the receptor (Abcam, IgG, Cat# ab3561, lot# GR45436-1) (dilution of 1:50) [43]; rabbit polyclonal anti-transferrin receptor (Abcam, IgG, Cat# ab84036) (dilution of 1:1000) [44]; rabbit polyclonal anti-flotillin-1 directed against residue surrounding Ile368 of human flotillin-1 (Cell Signaling, IgG, Cat# 3253) (dilution of 1:1000) [45]; mouse monoclonal anti-cytochrome C raised against amino acids 1–104 (Santa Cruz Biotechnology, IgG, Cat# sc-13156, lot# I1516) (dilution of 1:500) [46]; mouse monoclonal anti-stearoyl-CoA desaturase-1 (SCD1) (Abcam, IgG, Cat# ab19862, lot# GR138898-1) (dilution of 1:1000) [47]; rabbit polyclonal anti-caveolin-1 (Abcam, IgG, Cat# ab2910, lot# GR3382824-4) (dilution of 1:1000); mouse monoclonal anti-PrPc raised against N-terminal domain of PrPc (Abcam, IgG, Cat# ab61409, lot#GR3368463-1) (dilution of 1:1000) [48]. Anti-CB1 receptor antibody has been previously validated in our laboratory for western blot using both serial dilution and blocking peptide approaches to confirm the antibody’s specificity in rodent cortical tissue [49]. Anti-CB2 receptor antibody has been previously validated in Huang et al. [50] and Zhang et al. [51] for mouse lung and brain, respectively.
Animals
Juvenile male C57BL/6 (5–7 weeks old, n = 5) mice were used in the study. Animal care protocols and guidelines were approved by the University of Saskatchewan Animal Research Ethics Board, following the Canadian Council on Animal Care (protocol# 20130062). Mice were housed 3 per cage in standard polypropylene cages in a temperature-controlled (21°C) colony room on a 12/12-h light/dark cycle and provided with ad libitum access to food, water, and environmental enrichment. Animals were anesthetized using 5% isoflurane and cortical tissue was collected on ice.
Cell culture
The human epithelial mammary gland (MDA-MB-231) cell line was purchased from the American Type Culture Collection (ATCC, Cat# ATCC® HTB-26™) and cultured in the Roswell Park Memorial Institute 1640 medium (RPMI 1640, Thermofisher, Cat# 11875093) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich, Cat# F2442) and placed at 37 °C in a humidified incubator containing 5% CO2. Cells were grown to confluence in 100-mm dishes, washed in phosphate buffer saline (PBS) followed by isolation of PM compartments.
Isolation of lipid-rafts compartment by sucrose-gradient centrifugation
Detergent-resistant membrane (DRM) was isolated based on the standard protocol described previously [52–55]. Mouse cortical tissues or MDA-MB-231 cell line were lysed with cold tissue homogenization buffer [150 mM NaCl, 20 mM Na2HPO4, 2 mM NaH2PO4, 20% (v/v) glycerol, 2 mM sodium orthovanadate and protease inhibitors (Roche, Cat# 04 693 159 001, lot# 37536800), pH 7.4)] by 30 strokes in a Dounce homogenizer, followed by 20 passages through a 22-gauge needle. Then centrifuged for 11 min at 12,000×g in 4 °C to clear cellular debris and nuclear material. The supernatant was centrifuged at 124,000×g (SW55 rotor) for 90 min at 4 °C to pellet the total PM. The pellet was resuspended in 2 mL cold solubilizing buffer containing 0.5% v/v Triton X-100 in Mes-Buffered Saline (MBS, 25 mM MES, pH 6.5, 150 mM NaCl), protease inhibitors and 2 mM sodium orthovanadate and incubated for 30 min at 4 °C. Incubation time is important for this step to decrease contamination from other subcellular organelles, including, endoplasmic reticulum and mitochondria [56]. Two mL of solubilized PM was mixed with 2 mL of 80% (w/v) sucrose and applied to the bottom of a 12 mL ultracentrifuge tube. The 30% sucrose was layered on top, followed by 4 mL of MBS buffer containing 5% sucrose. The sucrose gradient was centrifuged at 164,000 xg (SW41Ti rotor) for 16 h at 4 °C to isolate the lipid raft and non-raft compartments. Twelve equal fractions (1 mL each) were collected from the top of gradient to the bottom. A creamy white layer at the 5–30% interface was identified and collected as lipid raft fraction.
Western blotting
Samples consisting an equal volume of each fraction were separated on 12% SDS-PAGE with the current in 90 V for 90 min and transferred onto polyvinylidene difluoride (PVDF) membranes (current 30 V, overnight). Membranes were incubated with 5% fat-free milk for 1 h at room temperature to block nonspecific background. The target proteins were immunoblotted with primary antibodies in 2% BSA overnight at 4 °C and then with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. The membranes were exposed to enhanced chemiluminescence reagent (Bio-Rad) and imaged using ChemiDoc™ MP Imaging System. Western blot images were analyzed using NIH ImageJ software.
Result
We evaluated CB1 and CB2 receptors distribution and abundance through sucrose density gradient centrifugation in the PM of mouse cortical tissue and MDA-MB-231 cell line. Resistance to non-ionic detergent extraction (Triton X-100) at 4 °C and association with detergent resistant membrane (DRM) is one of the main biochemical features of lipid rafts components [40]. After the isolation process, an intact lipid raft fraction floating on the top of the centrifuge tube, fractions 4 and 5, was visible in comparison to non-raft fractions, 8–12, at the bottom of the tube (Fig. 1). Western blot analysis on an equal volume aliquot of each fraction indicated that the distribution of CB1 receptor in the PM is associated with non-raft fractions (fractions 8–12) (Fig. 2A, B). Similarly, CB2 receptor was present in high-density membrane fractions (fractions 8–12) (Fig. 2A, B). Previous data demonstrate that western blot detection of GPCRs, including the CB receptors, results in the detection of multiple bands corresponding to isoform variants, potential dimers, and post-translations modifications [49, 51, 57]. In our study, only the bands for CB1 receptor (60 kDa) and CB2 receptor (40 kDa) were quantified. Experiments were performed with 5 independent samples to determine the mean ± S.E.M % of CB1 or CB2 receptor present within each obtained fraction (Fig. 2B). Immunoblot analysis of individual fractions indicated that the lipid raft marker, flotillin-1, caveolin-1 and PrPc was present in fraction 4 and 5, confirming the isolation of DRM. The non-raft marker, transferrin receptor, was localized in soluble fractions 8–12. We also verified that PM fractionation was relatively purified without endoplasmic reticulum and mitochondria contamination by blotting with SCD1 and Cytochrome C, as their protein markers, respectively (Fig. 2A).
Detergent-resistant microdomains were also isolated from MDA-MB-231 cells. We found that CB1 receptor in MDA-MB-231 cells was Triton X-100 insoluble and the majority of the receptor floated in the fractions 4 and 5 of the gradients. In contrast, CB2 receptor appeared almost completely restricted at detergent-soluble fractions 8–12. Flotillin-1 and caveolin-1 in fractions 4 and 5 demonstrated the lipid rafts compartment and transferrin receptors in fractions 8 to 12, represented non-raft (bulk membrane) compartments (Additional file 1: Figure S1).
Discussion
In the present study, we investigated the PM compartmental localization of CB1 and CB2 receptors in C57BL mouse cortical tissue and MDA-MB-231 cell line. In cortical tissue, both CB1 and CB2 receptors detected residing at the non-raft (bulk membrane compartments of PM). By contract, CB1 receptor enriched in lipid rafts of MDA-MB-231 cells, and CB2 receptor detected in non-lipid raft fractions.
The location of CB receptors in the PM helps to better understand the CB signalling pathway. The CB1 receptor cycles between the endosome and PM [58]. The role of lipid rafts/caveolae in compartmentalization of the endocannabinoid signalling machinery have been studied in several cultured cell systems [10]. These studies reported an association between the CB1 receptor with lipid raft/caveolae. In human embryonic kidney (HEK) 293 cells transfected with CB1 receptors, CB1 receptor internalization occurred, in parallel, through clathrin-coated pits and caveolae [59, 60]. CB1 receptor acylation at the C-terminal domain is necessary for proper interactions with lipid raft-associated G proteins [61–63]. In rat C6 glioma cells, the CB1 receptor co-localized with caveolin-1 [38]. Lipid raft disruption in rat C6 glioma cells by methyl-β-cyclodextrin (MCD) doubles CB1 receptor -dependent signaling through adenylyl cyclase and mitogen-activated protein kinases [64–66]. Moreover, in human breast cancer MDA-MB-231 cell line, the CB1 receptor associates with lipid raft/non-lipid raft fractions which relies on receptor activation/antagonism [67, 68]. CB1 receptor palmitoylation regulates membrane targeting and downstream signalling [69, 70]. Lipid pathway for ligand binding is essential for a CB receptor. Cholesterol acts as a CB1 receptor allosteric modulator [41]. These results point out that CB1 receptors are probably localized within lipid rafts [64, 66]. In contrast, in caveolin-1-lacking microglial cell line BV-2, only small amounts of CB1 receptor is found in lipid raft fractions [71]. Similar to our finding, the CB2 receptor has been shown to be localized in the non-lipid raft fraction of the dorsal root ganglion X neuroblastoma cell line (F-11) [72]. Proteins might translocate from non-raft to raft compartments through lateral movement along the PM. Different studies have shown that raft and non-raft segregation is a way to control protein function or determine protein destination. For example, the raft-localized neuronal Src is more active catalytically than in the soluble fraction [73]. Similarly, CB1 receptor -dependent signaling has been shown to be cell compartment specific in lipid rafts of HEK293 cells [60] and mitochondria [61]. In order to study this lateral movement of proteins, fluorescent molecular probes, which make lipid raft nanostructures visible through optical techniques can be helpful to find more details about the function of CB receptor in the PM. Further studies on CB receptors in PM are warranted to discover lateral movement for these receptors.
Our result from MDA-MB-231 cell line support previous studies related to the CB1 and CB2 receptors distribution in lipid raft and non-raft compartment respectively [67]. On the other hand, our findings indicate that CB1 and CB2 receptors are not co-localized with lipid rafts in mouse cortical tissue, which is in disagreement with the previous findings in cell lines. This inconsistency may arise from the difference between actual brain tissue used in our study with the mono-layer cell lines. Since mature neurones express less caveoline-1, it might decrease CB1 receptor association with lipid raft compartments of the cortical cell membrane. It has yet to be determined if caveolin-1 low expression affects the interaction of CB1 receptor with cortical tissue lipid rafts. This connection could explain why CB1 receptors are found in different PM compartments in different cell types. Moreover, The CB2 receptors were recovered in the higher density portion (non-rafts) of the gradient. It has been previously reported that membranes from different cells display altered average densities [40]. To avoid this average density alteration, we took advantage of using freshly dissected cortices. Our data describe the localization of CB receptors in juvenile, male, drug-naïve cortical tissue from mice and form a baseline for future research. These studies warrant more further investigations on PM compartmental distributions of CB1 and CB2 receptors in various brain anatomical sections.
Supplementary Information
Untitled section
Acknowledgements
Not applicable.
Funding
CT’s research was partly funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant Program (Grant Number 436192-2013). Funding for the project was provided by CIHR Partnership Grant with GlaxoSmithKline to RBL. This work was also supported by the University of Saskatchewan College of Pharmacy and Nutrition (RBL) and a student research scholarship to HB from the Colleges of Medicine and Pharmacy and Nutrition.
Availability of data and materials
Please contact author for data requests.
Declarations
Ethics approval and consent to participate
Animal care protocols and guidelines were approved by the University of Saskatchewan Animal Research Ethics Board (Protocol #: 20130062).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Footnotes
Footnote Group
Contributor Information
Robert B. Laprairie, Email: robert.laprairie@usask.ca
Changiz Taghibiglou, Email: changiz.taghibiglou@usask.ca.
References
Untitled section
References
- 1.Jahan P. Marcu JBS. Molecular pharmacology of CB1 and CB2 cannabinoid receptors. In: Preedy VR, editor. Neuropathology of drug addictions and substance misuse. Academic Press; 2016. p. 713-721.
- 2.Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci. 1991;11(2):563–583. doi: 10.1523/JNEUROSCI.11-02-00563.1991.
- 3.Kuner GM. Anatomical distribution of receptors, ligands and enzymes in the brain and in the spinal cord: circuitries and neurochemistry. In: Köfalvi A, editor. Cannabinoids and the brain. Boston: Springer US; 2008. pp. 161–201.
- 4.Mikasova L, Groc L, Choquet D, Manzoni OJ. Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization. Proc Natl Acad Sci U S A. 2008;105(47):18596–18601. doi: 10.1073/pnas.0805959105.
- 5.Howlett AC, Abood ME. CB(1) and CB(2) receptor pharmacology. Adv Pharmacol. 2017;80:169–206. doi: 10.1016/bs.apha.2017.03.007.
- 6.Klein TW. Cannabinoid-based drugs as anti-inflammatory therapeutics. Nat Rev Immunol. 2005;5(5):400–411. doi: 10.1038/nri1602.
- 7.Mackie K. Mechanisms of CB1 receptor signaling: endocannabinoid modulation of synaptic strength. Int J Obes (Lond) 2006;30(Suppl 1):S19–23. doi: 10.1038/sj.ijo.0803273.
- 8.Mackie K. Cannabinoid receptors: where they are and what they do. J Neuroendocrinol. 2008;20(Suppl 1):10–14. doi: 10.1111/j.1365-2826.2008.01671.x.
- 9.Palazuelos J, Aguado T, Pazos MR, Julien B, Carrasco C, Resel E, et al. Microglial CB2 cannabinoid receptors are neuroprotective in Huntington's disease excitotoxicity. Brain. 2009;132(Pt 11):3152–3164. doi: 10.1093/brain/awp239.
- 10.Maccarrone M, Bernardi G, Agro AF, Centonze D. Cannabinoid receptor signalling in neurodegenerative diseases: a potential role for membrane fluidity disturbance. Br J Pharmacol. 2011;163(7):1379–1390. doi: 10.1111/j.1476-5381.2011.01277.x.
- 11.Kendall DA, Yudowski GA. Cannabinoid receptors in the central nervous system: their signaling and roles in disease. Front Cell Neurosci. 2016;10:294. doi: 10.3389/fncel.2016.00294.
- 12.Rubino T, Zamberletti E, Parolaro D. Endocannabinoids and mental disorders. Handb Exp Pharmacol. 2015;231:261–283. doi: 10.1007/978-3-319-20825-1_9.
- 13.Wolf J, Urits I, Orhurhu V, Peck J, Orhurhu MS, Giacomazzi S, et al. The role of the cannabinoid system in pain control: basic and clinical implications. Curr Pain Headache Rep. 2020;24(7):35. doi: 10.1007/s11916-020-00873-9.
- 14.Vuckovic S, Srebro D, Vujovic KS, Vucetic C, Prostran M. Cannabinoids and pain: new insights from old molecules. Front Pharmacol. 2018;9:1259. doi: 10.3389/fphar.2018.01259.
- 15.Quarta C, Cota D. Anti-obesity therapy with peripheral CB1 blockers: from promise to safe(?) practice. Int J Obes. 2020;44(11):2179–2193. doi: 10.1038/s41366-020-0577-8.
- 16.Murphy T, Le Foll B. Targeting the endocannabinoid CB1 receptor to treat body weight disorders: a preclinical and clinical review of the therapeutic potential of past and present CB1 drugs. Biomolecules. 2020;10(6):855. doi: 10.3390/biom10060855.
- 17.Mecha M, Carrillo-Salinas FJ, Feliu A, Mestre L, Guaza C. Perspectives on cannabis-based therapy of multiple sclerosis: a mini-review. Front Cell Neurosci. 2020;14:34. doi: 10.3389/fncel.2020.00034.
- 18.Slaven M, Levine O. Cannabinoids for symptoms of multiple sclerosis: benefits to patients still unclear. JAMA Netw Open. 2018;1(6):e183484. doi: 10.1001/jamanetworkopen.2018.3484.
- 19.Le Foll B, Goldberg SR. Cannabinoid CB1 receptor antagonists as promising new medications for drug dependence. J Pharmacol Exp Ther. 2005;312(3):875–883. doi: 10.1124/jpet.104.077974.
- 20.Gamaleddin IH, Trigo JM, Gueye AB, Zvonok A, Makriyannis A, Goldberg SR, et al. Role of the endogenous cannabinoid system in nicotine addiction: novel insights. Front Psychiatry. 2015;6:41. doi: 10.3389/fpsyt.2015.00041.
- 21.Stampanoni Bassi M, Sancesario A, Morace R, Centonze D, Iezzi E. Cannabinoids in Parkinson's disease. Cannabis cannabinoid Res. 2017;2(1):21–29. doi: 10.1089/can.2017.0002.
- 22.More SV, Choi D-K. Promising cannabinoid-based therapies for Parkinson’s disease: motor symptoms to neuroprotection. Mol Neurodegener. 2015;10(1):17. doi: 10.1186/s13024-015-0012-0.
- 23.Liu CS, Chau SA, Ruthirakuhan M, Lanctôt KL, Herrmann N. Cannabinoids for the treatment of agitation and aggression in Alzheimer's disease. CNS Drugs. 2015;29(8):615–623. doi: 10.1007/s40263-015-0270-y.
- 24.Laprairie RB, Bagher AM, Kelly ME, Denovan-Wright EM. Biased type 1 cannabinoid receptor signaling influences neuronal viability in a cell culture model of Huntington disease. Mol Pharmacol. 2016;89(3):364–375. doi: 10.1124/mol.115.101980.
- 25.Turcotte C, Blanchet MR, Laviolette M, Flamand N. The CB2 receptor and its role as a regulator of inflammation. Cell Mol Life Sci. 2016;73(23):4449–4470. doi: 10.1007/s00018-016-2300-4.
- 26.Mukhopadhyay P, Baggelaar M, Erdelyi K, Cao Z, Cinar R, Fezza F, et al. The novel, orally available and peripherally restricted selective cannabinoid CB2 receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity. Br J Pharmacol. 2016;173(3):446–458. doi: 10.1111/bph.13338.
- 27.Turu G, Hunyady L. Signal transduction of the CB1 cannabinoid receptor. J Mol Endocrinol. 2010;44(2):75–85. doi: 10.1677/JME-08-0190.
- 28.Martin BR. Cellular effects of cannabinoids. Pharmacol Rev. 1986;38(1):45–74.
- 29.Brailoiu GC, Oprea TI, Zhao P, Abood ME, Brailoiu E. Intracellular cannabinoid type 1 (CB1) receptors are activated by anandamide. J Biol Chem. 2011;286(33):29166–29174. doi: 10.1074/jbc.M110.217463.
- 30.Bénard G, Massa F, Puente N, Lourenço J, Bellocchio L, Soria-Gómez E, et al. Mitochondrial CB1 receptors regulate neuronal energy metabolism. Nat Neurosci. 2012;15(4):558–564. doi: 10.1038/nn.3053.
- 31.Brusco A, Tagliaferro P, Saez T, Onaivi ES. Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus. Synapse. 2008;62(12):944–949. doi: 10.1002/syn.20569.
- 32.den Boon FS, Chameau P, Schaafsma-Zhao Q, van Aken W, Bari M, Oddi S, et al. Excitability of prefrontal cortical pyramidal neurons is modulated by activation of intracellular type-2 cannabinoid receptors. Proc Natl Acad Sci U S A. 2012;109(9):3534–3539. doi: 10.1073/pnas.1118167109.
- 33.Brailoiu GC, Deliu E, Marcu J, Hoffman NE, Console-Bram L, Zhao P, et al. Differential activation of intracellular versus plasmalemmal CB2 cannabinoid receptors. Biochemistry. 2014;53(30):4990–4999. doi: 10.1021/bi500632a.
- 34.Gaus K, Gratton E, Kable EP, Jones AS, Gelissen I, Kritharides L, et al. Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci U S A. 2003;100(26):15554–15559. doi: 10.1073/pnas.2534386100.
- 35.Wilson BS, Steinberg SL, Liederman K, Pfeiffer JR, Surviladze Z, Zhang J, et al. Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. Mol Biol Cell. 2004;15(6):2580–2592. doi: 10.1091/mbc.e03-08-0574.
- 36.Levental I, Veatch S. The continuing mystery of lipid rafts. J Mol Biol. 2016;428(24 Pt A):4749–4764. doi: 10.1016/j.jmb.2016.08.022.
- 37.Ouweneel AB, Thomas MJ, Sorci-Thomas MG. The ins and outs of lipid rafts: functions in intracellular cholesterol homeostasis, microparticles, and cell membranes. J Lipid Res. 2020;61(5):676–686. doi: 10.1194/jlr.TR119000383.
- 38.Bari M, Oddi S, De Simone C, Spagnolo P, Gasperi V, Battista N, et al. Type-1 cannabinoid receptors colocalize with caveolin-1 in neuronal cells. Neuropharmacology. 2008;54(1):45–50. doi: 10.1016/j.neuropharm.2007.06.030.
- 39.Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992;68(3):533–544. doi: 10.1016/0092-8674(92)90189-J.
- 40.Macdonald JL, Pike LJ. A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res. 2005;46(5):1061–1067. doi: 10.1194/jlr.D400041-JLR200.
- 41.Hua T, Li X, Wu L, Iliopoulos-Tsoutsouvas C, Wang Y, Wu M, et al. Activation and signaling mechanism revealed by cannabinoid receptor-Gi complex structures. Cell. 2020;180(4):655–65 e18. doi: 10.1016/j.cell.2020.01.008.
- 42.Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience. 1998;83(2):393–411. doi: 10.1016/S0306-4522(97)00436-3.
- 43.Gupta B, Hornick MG, Briyal S, Donovan R, Prazad P, Gulati A. Anti-apoptotic and immunomodulatory effect of CB2 agonist, JWH133, in a neonatal rat model of hypoxic-ischemic encephalopathy. Front Pediatr. 2020;8:65. doi: 10.3389/fped.2020.00065.
- 44.Song J, Ping LY, Duong DM, Gao XY, He CY, Wei L, et al. Native low density lipoprotein promotes lipid raft formation in macrophages. Mol Med Rep. 2016;13(3):2087–2093. doi: 10.3892/mmr.2016.4781.
- 45.Griner LN, McGraw KL, Johnson JO, List AF, Reuther GW. JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth. Br J Haematol. 2013;160(2):177–187. doi: 10.1111/bjh.12103.
- 46.An X, Ji B, Sun D. TRIM34 localizes to the mitochondria and mediates apoptosis through the mitochondrial pathway in HEK293T cells. Heliyon. 2020;6(1):e03115. doi: 10.1016/j.heliyon.2019.e03115.
- 47.Peter A, Weigert C, Staiger H, Machicao F, Schick F, Machann J, et al. Individual stearoyl-coa desaturase 1 expression modulates endoplasmic reticulum stress and inflammation in human myotubes and is associated with skeletal muscle lipid storage and insulin sensitivity in vivo. Diabetes. 2009;58(8):1757–1765. doi: 10.2337/db09-0188.
- 48.Caputo A, Sarnataro D, Campana V, Costanzo M, Negro A, Sorgato MC, et al. Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells. Biochem J. 2009;425(2):341–351. doi: 10.1042/BJ20091050.
- 49.Roebuck AJ, Greba Q, Smolyakova AM, Alaverdashvili M, Marks WN, Garai S, Baglot SL, Petrie G, Cain SM, Snutch TP, Thakur GA, Howland JG, Laprairie RB. Positive allosteric modulation of type 1 cannabinoid receptors reduces spike-and-wave discharges in genetic absence epilepsy rats from Strasbourg. Neuropharmacology. 2021;190(1):108553. doi: 10.1016/j.neuropharm.2021.108553.
- 50.Huang W, Xiong Y, Chen Y, Cheng Y, Wang R. NOX2 is involved in CB2-mediated protection against lung ischemia-reperfusion injury in mice. Int J Clin Exp Pathol. 2020;13(2):277–285.
- 51.Zhang K, Yang Q, Yang L, Li YJ, Wang XS, Li YJ, et al. CB1 agonism prolongs therapeutic window for hormone replacement in ovariectomized mice. J Clin Invest. 2019;129(6):2333–2350. doi: 10.1172/JCI123689.
- 52.Taghibiglou C, Bradley CA, Gaertner T, Li Y, Wang Y, Wang YT. Mechanisms involved in cholesterol-induced neuronal insulin resistance. Neuropharmacology. 2009;57(3):268–276. doi: 10.1016/j.neuropharm.2009.05.013.
- 53.Li X, Serwanski DR, Miralles CP, Bahr BA, De Blas AL. Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex. J Neurochem. 2007;102(4):1329–1345. doi: 10.1111/j.1471-4159.2007.04635.x.
- 54.Nothdurfter C, Tanasic S, Di Benedetto B, Uhr M, Wagner EM, Gilling KE, et al. Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds. Int J Neuropsychopharmacol. 2013;16(6):1361–1371. doi: 10.1017/S146114571200140X.
- 55.Gil C, Dorca-Arevalo J, Blasi J. Clostridium perfringens epsilon toxin binds to membrane lipids and its cytotoxic action depends on sulfatide. PLoS ONE. 2015;10(10):e0140321. doi: 10.1371/journal.pone.0140321.
- 56.Kim KB, Lee JS, Ko YG. The isolation of detergent-resistant lipid rafts for two-dimensional electrophoresis. Methods Mol Biol. 2008;424:413–422. doi: 10.1007/978-1-60327-064-9_32.
- 57.Bagher AM, Laprairie RB, Kelly ME, Denovan-Wright EM. Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes. Eur J Pharmacol. 2013;721(1–3):341–354. doi: 10.1016/j.ejphar.2013.09.002.
- 58.Leterrier C, Bonnard D, Carrel D, Rossier J, Lenkei Z. Constitutive endocytic cycle of the CB1 cannabinoid receptor. J Biol Chem. 2004;279(34):36013–36021. doi: 10.1074/jbc.M403990200.
- 59.Keren O, Sarne Y. Multiple mechanisms of CB1 cannabinoid receptors regulation. Brain Res. 2003;980(2):197–205. doi: 10.1016/S0006-8993(03)02970-6.
- 60.Wu DF, Yang LQ, Goschke A, Stumm R, Brandenburg LO, Liang YJ, et al. Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors. J Neurochem. 2008;104(4):1132–1143. doi: 10.1111/j.1471-4159.2007.05063.x.
- 61.Mukhopadhyay S, Cowsik SM, Lynn AM, Welsh WJ, Howlett AC. Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis. Biochemistry. 1999;38(11):3447–3455. doi: 10.1021/bi981767v.
- 62.Barnett-Norris J, Lynch D, Reggio PH. Lipids, lipid rafts and caveolae: their importance for GPCR signaling and their centrality to the endocannabinoid system. Life Sci. 2005;77(14):1625–1639. doi: 10.1016/j.lfs.2005.05.040.
- 63.Xie XQ, Chen JZ. NMR structural comparison of the cytoplasmic juxtamembrane domains of G-protein-coupled CB1 and CB2 receptors in membrane mimetic dodecylphosphocholine micelles. J Biol Chem. 2005;280(5):3605–3612. doi: 10.1074/jbc.M410294200.
- 64.Bari M, Battista N, Fezza F, Finazzi-Agro A, Maccarrone M. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apoptosis. J Biol Chem. 2005;280(13):12212–12220. doi: 10.1074/jbc.M411642200.
- 65.Bari M, Paradisi A, Pasquariello N, Maccarrone M. Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells. J Neurosci Res. 2005;81(2):275–283. doi: 10.1002/jnr.20546.
- 66.Bari M, Spagnuolo P, Fezza F, Oddi S, Pasquariello N, Finazzi-Agro A, et al. Effect of lipid rafts on Cb2 receptor signaling and 2-arachidonoyl-glycerol metabolism in human immune cells. J Immunol. 2006;177(8):4971–4980. doi: 10.4049/jimmunol.177.8.4971.
- 67.Sarnataro D, Grimaldi C, Pisanti S, Gazzerro P, Laezza C, Zurzolo C, et al. Plasma membrane and lysosomal localization of CB1 cannabinoid receptor are dependent on lipid rafts and regulated by anandamide in human breast cancer cells. FEBS Lett. 2005;579(28):6343–6349. doi: 10.1016/j.febslet.2005.10.016.
- 68.Sarnataro D, Pisanti S, Santoro A, Gazzerro P, Malfitano AM, Laezza C, et al. The cannabinoid CB1 receptor antagonist rimonabant (SR141716) inhibits human breast cancer cell proliferation through a lipid raft-mediated mechanism. Mol Pharmacol. 2006;70(4):1298–1306. doi: 10.1124/mol.106.025601.
- 69.Oddi S, Dainese E, Sandiford S, Fezza F, Lanuti M, Chiurchiu V, et al. Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling. Br J Pharmacol. 2012;165(8):2635–2651. doi: 10.1111/j.1476-5381.2011.01658.x.
- 70.Oddi S, Stepniewski TM, Totaro A, Selent J, Scipioni L, Dufrusine B, et al. Palmitoylation of cysteine 415 of CB1 receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1. Biochim Biophys Acta Mol Cell Biol Lipids. 2017;1862(5):523–532. doi: 10.1016/j.bbalip.2017.02.004.
- 71.Rimmerman N, Bradshaw HB, Kozela E, Levy R, Juknat A, Vogel Z. Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1. Br J Pharmacol. 2012;165(8):2436–2449. doi: 10.1111/j.1476-5381.2011.01380.x.
- 72.Rimmerman N, Hughes HV, Bradshaw HB, Pazos MX, Mackie K, Prieto AL, et al. Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line. Br J Pharmacol. 2008;153(2):380–389. doi: 10.1038/sj.bjp.0707561.
- 73.Mukherjee A, Arnaud L, Cooper JA. Lipid-dependent recruitment of neuronal Src to lipid rafts in the brain. J Biol Chem. 2003;278(42):40806–40814. doi: 10.1074/jbc.M306440200.
Associated Data
Supplementary Materials
Data Availability Statement
Please contact author for data requests.