Receptors of fatty acids and endocannabinoids; lipid rafts

1. Receptors of fatty acidsand endocannabinoids;lipid rafts mirka.rovenska@lfmotol.cuni.cz

2. Receptors of fatty acids • 1) Peroxisomal proliferator-activated receptors(PPAR) • nuclear • 2) Free-fatty acid-activated receptors(FFAR) • on the cell surface, coupled to G proteins

3. (oxidized LDL) (PPAR responsive element) The PPAR family of receptors • 3 isoforms: • PPAR • PPARδ/ • PPARγ • Regulation of lipid metabo-lism: they bind to response elements to stimulate transcription of FA oxidation and lipid synthesis genes • They bind to DNA as hetero-dimers with the retinoid X receptor (RXR, activated by 9-cis retinoic acid)

4. Ligands of PPARs S y n t h e t i c N a t u r a l • Major natural ligands: free fatty acids with long chains(> 12C), particularly poly-unsaturated FAs HODE=hydroxyoctadecadienoic acid

5. Effects of PPAR • PPARαregulates expression of various genes implicated in lipid oxidation, mainly in the liver and oxidative muscles, such as the heart • PPARγ is involved in lipogenesis and terminal differentiation of adipocytes in white adipose tissue

6. PPAR • Expression is high in the tissues with high rates of FA oxidation: liver, heart, skeletal muscle, kidney, brown fat • Function: regulation of the • cellular uptake • activation • β-oxidationof FA: stimulates the peroxisomalβ-oxidation pathway profoundly, mitochondrial to a lesser extent • PPAR expression is upregulated during fasting and stress (i.e. when FAs are released from the adipose tissue) • Lipid-lowering drugs of the fibrate class (bezafibrate) are potent activators of PPAR

7. PPARα target genes • LPL • fatty acid transport protein (FATP), fatty acid translocase (FAT) • acyl-CoA synthetase • enzymes of peroxisomal β-oxidation (acyl-CoA oxidase, thiolase) • enzymes involved in mitochondrial β-oxidation (CPT1, acyl-CoA dehydrogenase) • enzymes of ketogenesis: 3-hydroxy-3-methylglutaryl-CoA synthase

8. PPAR/δ • Ubiquitous; high expression in brain, adipose tissue, skin • Particularly abundant during development (such as in CNS) • Suggested to be involved in the differentiation of cells within the CNS, myelinization, and lipid metabolism in brain • May be also implicated in basiccellular functions, such as membrane lipid synthesisand turnover • Similarly to PPAR, it stimulates the expression of proteins of FA oxidation (heart)

9. PPARγ • 3 isoforms: • PPARγ1 – in wide range of tissues • PPARγ2 – predominantly in adipose tissue • PPARγ3 – only in adipose tissue, macrophages, colon • Functions: • regulation of adipocyte differentiation • regulation of lipid anabolism: in the adipose tissue, they regulatethe expression of LPL, FATP… • PPARγ are targeted by anti-diabetic substances of the thiazolidine-dione class (glitazones, e.g.rosiglitazone) that enhance the tissue sensitivity to insulin and reduce the plasma levels of FA and Glc

10. The FFAR family of receptors • Localized to the plasma membrane (unlike PPARs) and coupled to G proteins • Ligand spectrum includes FA chain lengths down to one (unlike PPARs) • 3 isoforms: • FFA1R • FFA2R • FFA3R

11. Roles of FFARs

12. Endocannabinoids • Biologically active lipophilic substances that activate cannabinoid receptors • Derivatives of arachidonic acid, which are generated from membrane phospholipids in response to stimuli • Two best-characterized:

13. Synthesis of anandamide • The reaction is initiated by activating neurotransmitter receptors and/or by elevated intracellular Ca2+ (PE)

14. Synthesis of 2-arachidonoylglycerol • Also linked to elevated intracellular Ca2+ • 2 pathways: • phospholipase C + diacylglycerol lipase • phospholipase A1 + phospholipase C (specific for lyso-PI) a) b)

15. Function • Paracrine mediators (rapidly eliminated through uptake into cells and enzymatic hydrolysis) • Produced mainly in the: • nervous system • immune system • Anandamide levels in tissues – very low and in some cases, it does not act as a full agonist  physiological significance questionable • On the other hand, 2-AG is a full agonist at the CB1 as well as CB2 receptor and the levels in tissues are much higher

16. Cannabinoid receptors • Targeted by THC (Δ9-tetrahydrocannabinol) • CB1 – most abundant in the CNS, also present in immune cells, lung, small intestine, uterus, testis… • CB2 – found mostly in the immune system (leukocytes, spleen, lymph nodes) • 7-transmembrane, coupled to Gi/Go proteins inhibition of adenylyl cyclase… THC

17. Physiological roles • 2-AG from stimulated neurons attenuates neurotransmitter release by decreasing intracellular Ca2+ (?calming the excitation of neurons to prevent cell death?) • 2-AG suppresses long-term potentiation • ?Role in immune response? (CB2 – in the immune system) • CB1 stimulation inhibits proliferation of human breast cancer cells

18. Lipid rafts Plasma membrane: NOT absolutely homogeneous!

19. Lipid rafts • Sphingolipid- and cholesterol-rich microdomains of the plasma membrane containing a variety of signalling and transport proteins • Resistant to mild detergents • Highly dynamic, densely organized • This environment fits for transport, conformational changes of signal transducers, but also for pathogen entry into the host cell (HIV, Ebola) (10-200 nm)

20. A model of a lipid raft • Cylindrical glycero-phospholipids (GPLs) form the disordered Lc phase of the membrane • Ordered Lo phase – raft: in the outer leaflet, cholesterol fills the voids between sphingolipids (SM); in the inner leaflet, chol. fills the voids between selected (pyramidal) GPLs • This organization rigidifies the membrane

21. Proteins in lipid rafts • Raft resident proteins are often GPI(glycosylphosphatidylinositol) anchored: • Rafts contain: • receptors (Fas, MHCI, II) • signalling molecules(tyrosin kinases, Gproteins) • transporters (GLUT4, FAT)

22. Lipid rafts facilitate TCR-mediated T cell activation • TCR moves to rafts during T-cell activation • Formation of the TCR-MHC complexes and aggregation of co-stimulatory molecules in lipid rafts, raft aggregation • Tyr phosphorylation and recruitment of signalling proteins

23. Caveolae • Types of rafts that are rich in proteins of the caveolin family (caveolin-1,-2,-3) • Caveolin-1, integral membrane protein, forms oligomers that associate with each other and form a pit in the membrane • Proposed roles: • signalling • transport • pathogen entry (SV40, E. Coli)

24. Raft-mediated HIV entry into the host cell – a model • gp120 (viral), CD4, and sphingolipids (SLs) form a complex in the raft area; SLs stabilize HIV on the cell surface • The raft floats on the cell surface to the co-receptor for the CD4–gp120 complex • SLs facilitate the conformatio-nal changes of gp120 that lead to the shedding of gp120 → release of the N-terminus of viral gp41 (initially buried in a gp120 pocket) • This „fusion peptide“ penetra-tes into the plasma membrane

25. Rafts in prion infection • The interaction of PrPC with lipid rafts (sphingolipids) might stabilize the ‘normal’ conformation of PrPC • These interactions should be destabilized when exogenous (shed from an infected cell) PrPSc is inserted in the vicinity of PrPC • Formation of PrPC/PrPSc complex (probably by coalescence of both rafts), PrPC → PrPSc conversion • Propagation on the cell surface