Receptors of fatty acids eicosanoids and endocannabinoids lipid

Receptors of fatty acids, eicosanoids, and endocannabinoids; lipid rafts mirka. rovenska@lfmotol. cuni. cz

Receptors of fatty acids Ø 1) Peroxisomal proliferator-activated receptors (PPARs) – nuclear Ø 2) Free-fatty acid-activated receptors (FFARs) – on the cell surface, coupled to G proteins

The PPAR family of receptors n Ø Ø Ø n n n 3 isoforms: PPARδ (sometimes called PPAR ) PPARγ They regulate lipid metabolism: function as transcription factors – bind to various response elements to stimulate transcription of FA oxidation and lipid synthesis genes They bind to DNA as heterodimers together with the retinoid X receptor (RXR, activated by 9 -cis retinoic acid) Major natural ligands: free fatty acids with long carbon chains (above 12 C), particularly polyunsaturated FAs

Ligands of PPARs Synthetic Natural (oxidized LDL) transcription (PPAR responsive element) (HODE, hydroxyoctadecadienoic acid)

Effects of PPARs n Ø Ø Ø PPARs control the expression of: FAT/CD 36 FA transport into the cell FABP muscle CPT-I acyl-Co. A dehydrogenases LPL ACC

PPAR n Expression is high in the tissues with high rates of FA oxidation: liver, heart, skeletal muscle, kidney, brown fat n Function: regulation of the cellular uptake activation and -oxidation of FA: stimulates the peroxisomal -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 Ø Ø Ø n n n Suggested to be involved in immunomodulation

PPAR /δ n Ubiquitous; particularly high expression in brain, adipose tissue, skin n Particularly abundant during development n Suggested to be involved in the differentiation of cells within the CNS, myelinization, and lipid metabolism in brain n Important for wound healing n Similarly to PPAR , it stimulates the expression of proteins of FA oxidation (heart)

PPARγ n Ø Ø Ø 3 isoforms: PPARγ 1 – in wide range of tissues PPARγ 2 – predominantly in adipose tissue PPARγ 3 – only in adipose tissue, macrophages, colon Functions: regulation of adipocyte differentiation lipid anabolism (in adipose tissue, it induces the expression of proteins involved in lipid synthesis– LPL, CD 36) are targeted by anti-diabetic substances of the thiazolidinedione class (glitazones, e. g. rosiglitazone) that enhance the sensitivity of the tissue to insulin and reduce the plasma levels of FA and Glc

The FFAR family of receptors n n n Ø Ø Ø 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: FFA 1 R FFA 2 R FFA 3 R

Roles of FFARs FFA 1 R Expression pancreatic islets, liver, skeletal muscle, heart FFA 2 R monocytes, neutrophils Ligands medium- (down to 10 C) short-chain FAs to long-chain FAs (C 1 -C 6) Effects stimulation of insulin secretion FFA 3 R adipose tissue, pancreas, immune cells similar to FFA 2 R mobilization of control of Ca 2+, arrest of leptin chemotaxis production (control of the invasion of immune cells in GIT)

Eicosanoid receptors n n A) G protein-coupled receptors B) Probably also nuclear receptors (PPAR) in case of distinct eicosanoids (PGJ, PGI 2) Leukotriene receptors BLT Cys. LT Lipoxin receptor ALX Prostaglandin receptors Peripheral blood leukocytes Peripheral blood Neutrophils, lung, Multiple PGRs often leukocytes, spleen co-expressed in a lung single cell type Highest affinity for LTB 4 High affinity for LTC 4, LTD 4 Receptor of LXA 4 EP for PGE, similarly FP, DP, IP chemotaxis Mediator in asthma antagonists (monteleukast) used clinically Mediates the antiinflammatory effects of LXA 4 The effect depends on what receptors and PGs are present in the given tissue

Endocannabinoids n n n 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:

Synthesis of anandamide (PE) n The reaction is initiated by activating neurotransmitter receptors and/or by elevated intracellular Ca 2+

Synthesis of 2 -arachidonoylglycerol n a) b) n Ø Ø Also linked to intracellular Ca 2+ rises 2 pathways: a) phospholipase C + diacylglycerol lipase b) phospholipase A 1 + phospholipase C (specific for lyso-PI)

Function n Paracrine mediators (rapidly eliminated through uptake into cells and enzymatic hydrolysis) Produced mainly in the: Ø nervous system Ø immune system n

Cannabinoid receptors THC n Targeted by THC (Δ 9 -tetrahydrocannabinol) Ø CB 1 – most abundant in the CNS, also present in immune cells, lung, small intestine, uterus, testis… CB 2 – found mostly in the immune system (leukocytes, spleen, lymph nodes) Ø n 7 -transmembrane, coupled to Gi/Go proteins initiate inhibition of adenylyl cyclase, opening of K+ channels, closing of Ca 2+ channels, stimulation of protein kinases

n Anandamide levels in tissues – very low and in some cases, it does not act as a full agonist its physiological significance remains questionable X n On the other hand, 2 -AG is a full agonist at the CB 1 as well as CB 2 receptor and the levels in tissues are much higher are CB 1 and CB 2 primarily 2 -AG receptors?

Physiological roles n n n 2 -AG from stimulated neurons attenuates neurotransmitter release by decreasing intracellular Ca 2+ (? calming the excitation of neurons to prevent cell death? ) 2 -AG suppresses long-term potentiation 2 -AG induces relaxation of blood vessels in vitro ? Role in immune responses? Anandamide was shown to elicit immobility, analgesia, inhibition of neurotransmitter release, impairment of memory, vasodilatation. BUT: anandamide binds also to other receptors that might mediate these effects CB 1 stimulation inhibits proliferation of human breast cancer cells

Lipid rafts Plasma membrane: NOT absolutely homogeneous!

Lipid rafts n n (10 -200 nm) n Sphingolipid- and cholesterolrich microdomains of the plasma membrane which contain 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, cholera toxin)

A model of a lipid raft n Cylindrical glycerophospholipids (GPLs) form the disordered Lc phase of the membrane n 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 n This organization rigidifies the membrane

Proteins in lipid rafts n Raft resident proteins are often GPI (glycosylphosphatidylinositol) anchored: n Rafts contain: receptors (Fas, CB 1, MHCI and II of APC) signalling molecules – tyrosin kinases (Src), G proteins transporters (GLUT 4, FAT/CD 36) Ø Ø Ø

Lipid rafts facilitate TCR-mediated T cell activation n TCR moves to rafts during T-cell activation n Formation of the TCR MHC complexes and aggregation of costimulatory molecules in lipid rafts, raft aggregation n Tyr phosphorylation and recruitment of signalling proteins

Raft-mediated HIV entry into the host cell – a model n n gp 120 (viral), CD 4, 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 CD 4–gp 120 complex SLs facilitate the conformational changes of gp 120 that lead to the shedding of gp 120 → release of the N-terminus of viral gp 41 (initially buried in a gp 120 pocket) This „fusion peptide“ penetrates into the plasma membrane of the target cell

Rafts in prion infection n n n The interaction of Pr. PC with lipid rafts (sphingolipids) might stabilize the ‘normal’ conformation of Pr. PC These interactions should be destabilized when exogenous (shed from an infected cell) Pr. PSc is inserted in the vicinity of Pr. PC Formation of Pr. PC/Pr. PSc/coreceptor complex (probably by coalescence of both rafts) Pr. PC → Pr. PSc conversion Propagation on the cell surface Formation of fibrils that accumulate in the brain

Caveolae n n n Ø Ø Ø 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 cholesterol transport pathogen entry (SV 40, some E. Coli)

Caveolae