Regulation of the metabolism PRINCIPLESOF THE REGULATION OF THE METABOLISM: THEORETIC BASES ENZYMES -BIOCATALYSATORS HORMONES(Common mechanisms of the effect of hormones and neurotransmiters) RECEPTORS (Type of membrane receptors and intracelularreceptors) ENZYMES VITAMINS METABOLIC REGULATIONS pathobiochemistry- receptors_1 6 General principles  Higher organisms, from the fruit fly to humans, are comprised of cells.  The cells often unite to form tissue which come together to form organs which together make up the organism.  Cells of an organism do not live in isolation.  The communication between cells ultimately controls growth, differentiation, and metabolic processes within the organism.  Communication between cells is often by direct cell to cell contact.  Communication frequently occurs between cells over short and long distances. Hormones, Receptors, and Signal Transduction pathobiochemistry- receptors_1 7 General principles cont...  In cases of short and long distance communication, a substance may be released by one cell and recognized by a different target cell.  In the target cell, a specific response is induced.  Cells use an amazing number of signaling chemicals.  These signaling molecules are termed “hormones.”  The ability of a hormone to induce a response in a target cell is usually mediated by a hormone receptor on, or in, the target cell. pathobiochemistry- receptors_1 8 General characteristics of hormones  Hormones are molecules synthesized by specific tissue. Classically these tissue were called glands.  Hormones are secreted directly into the blood which carries them to their sites of action.  Hormones are present at very low levels in the circulatory system.  Hormones specifically affect or alter the activities of the responsive tissue (target tissue).  Hormones act specifically via receptors located on, or in, target tissue. pathobiochemistry- receptors_1 9 Hormone/Receptor Interaction Secondary Signals pathobiochemistry- receptors_1 10 Hormones Reproduction Growth & Development Maintenance of internal environment Energy production, utilization & storage The four primary arenas of hormone action pathobiochemistry- receptors_1 11 Definitions Endocrine - Refers to the internal secretion of biologically active substances. Exocrine - Refers to secretion outside the body, for example, through sweat glands, mammary glands, or ducts lead to the gastrointestinal. Hormone - Substances released by an endocrine gland and transported through the bloodstream to another tissue where it acts to regulate functions in the target tissue (classic definition). Paracrine - Hormones that act locally on cells that did not produce them. Autocrine - Hormones that act on cells that produced them. Receptors -Hormones bind to receptors molecules on cells. A receptor must specifically recognize the hormone from the numerous other molecules in the blood and transmit the hormone binding information into a cellular specific action. pathobiochemistry- receptors_1 12 TEST Endocrine Blood vessel Distant target cells Hormone secretion into blood by endocrine gland Paracrine Secretory cell Adjacent target cell Autocrine Target sites on same cell Receptor Hormone or other extra cellular signal pathobiochemistry- receptors_1 13 pathobiochemistry- receptors_1 14 Effects of signal molecules Name of the effect Character of the effect endocrine The signal molecule is carried by blood into the target cell, which is usually distant from the place of the synthesis. Typically hormones paracrine The signal molecule is secreted into the ambient surroundings of the cell(local mediators). The signal molecule influences only the cells of the nearest surroundings. autocrine The cell secretes the signal molecule and it is also a target. Features ale similer like paracrine effect. Endocrine– the signal molecule is carried by blood into the target cell , which is usually distant from the place of the synthesis. It is typical for hormones. The concentration of the signal molecule in blood is very low (10−12–10−9 mol/l) – so target cell has a big affinity to the signal molecule – the binding hormone to receptor is very strong, hormone doesn´tdissociate easy. Other feature is that it take definite time that the concentration of the hormone in blood will rise and the concentration of the homone in blood stays for definite time (a few minute or hour) rised. Paracrine–Thesignal molecule is secreted into the ambient surroundings of the cell (local mediators). The signal molecule influences only the cells of the nearest surroundings. The concenration of the signal molecule in the surroundings of the cells is higher (10−9–10−6 mol/l). Affinity of the receptors to the signal molecule is lower – after decrease of the concentratrion in the surroundings of the cell, the signal molecule is separated. Paracrine signaling is determined for fast and localised communication between cells. Autocrine –The cell secretes the signal molecule and it is also a target. Features ale similer like paracrine effect. Juxtacrine– signalling between cells or cell and extracellular matrix require tight contact. TEST pathobiochemistry- receptors_1 15 Common mechanisms of the effect of hormones and neurotransmiters. Types signal molecules in the neurohumoral regulations: Signal molecule Source HORMONES secreted by endocrine glands, scattered glandular cells, eicosanoids a lot of other types of cells NEUROHORMONES secreted by neurons into the blood circulation NEUROTRANSMITERS secreted in the synaptic ending CYTOKINES,GROWTH FACTORS, EICOSANOIDS secreted by a lot of types of cells, usually not from endocrine glands TEST 16 Regulation of the metabolism is in the different levels, but always on the molecular base The Regulation of enzymatic reactions is a central instrument of the regulation of the metabolism. Regulation in the definite cellular compartment Regulation in the complete cell (proteome, specific receptors, izoenzymes, transporters, energetic state of the cell) Regulations followed from the communication between cells Levels of the regulation overlap. pathobiochemistry- receptors_1 17 Regulation of the enzymatic activity • regulation of the amount of enzyme (synthesis and degradation) • regulation of the activity of the enzyme (modification of the enzyme by proteolysis, covalent modification, allosteric regulation, interaction with the regulatory proteins) • availability and concentration of the substrate (regulation of the transport) pathobiochemistry- receptors_1 18 The collective feature of all substances with modulating effects to the cells is their effect by receptors. Receptors are allosteric proteins, which change their conformation after the bindind ligand. Ligands are signal molecules. Agonists are ligands, where after the binding on the receptor cause the transduction of signal, antagonists after the binding on the receptor defend the signal transduction. Receptors are localised on the outer surface of the cytoplazmatic membrane or intracellulary. In their structure there are two main components: (1) domain binding the ligand, which ensures the specifity of the binding with the relevant ligand; (2) efector domain, wich start a genesis of the biological answer after the binding of the ligand. Activated receptor can enter into the reaction with other cellular components and realise the process of the signal transduction. Tissues, whose cells have no molecules of the specific receptor, can´t react to the relevant hormone. Charakteristic feature of a transport of the signal by receptors is its amplification, when the only one molecule of the hormone is able to cause celullar answer with 104–105 times higher intensity. TEST pathobiochemistry- receptors_1 19 Principle of hierarchy in some hormonal regulations and amplification of the flow of information by signal molecules Neurons of the brain cortex NEUROTRANSMITERERS the highest nanograms Neurons of nucleuses of the hypothalamus CORTICOLIBERIN micrograms Cells of the adenohypophysis KORTICOTROPIN hundredsof micrograms Cells of the cortex of the adrenal glands CORTISOL hundreds of micrograms TARGET CELLS OF PERIPHERALTISSUES Dailyproduction:For example. pathobiochemistry- receptors_1 20 Signal transduction How does the cell take over the information carried by the chemical signal? Reaction of the signal molecule with the receptor Membrane receptors Proteins and smaller signal moleculs (peptides, amino acids, biogenic amins, eicosanoids) Intracelular receptors Nepolarsignal molecules (steroids, jodthyronins, retinoats) TEST pathobiochemistry- receptors_1 21 Amplification Membrane and intracelular receptors Interaction of the complex hormone- receptor with hormonsensitive element of DNA Nonpolar signaling molecules bound to plasma transportprotein Intracellular receptor Biological effect (slower effect) Polar signaling molecules Biological effect (fast effect, may be followed by belated action) Transduction of signal Membrane receptor Transport of signal molecule 1 100 10 000 TEST Receptors Cell surface membrane receptors Polypeptidehormonesand catecholamines Cytoplasmic receptors Most steroid and thyroid hormones Nuclear receptors estrogens pathobiochemistry- receptors_1 22 Secondary Messenger or Secondary Signal Cellular Trafficking Enzymes Activated Inhibited Nucleus DNA Synthesis RNA Synthesis Protein Synthesis Membrane Effects Receptor Effector Plasma Membrane Hormone A general model for the action of peptide hormones, catecholamines, and other membrane-active hormones. The hormone in the extra cellular fluid binds to the receptor and activates associated effector(s) systems, that may or may not be in the same molecule. This activation results in generation of an intracellular signal or second messenger that, through a variety of common and branched pathways, produces the final effects of the hormone on metabolic enzyme activity, protein synthesis, or cellular growth and differentiation. pathobiochemistry- receptors_1 23 pathobiochemistry- receptors_1 24 pathobiochemistry- receptors_1 25 Types of receptors Type of receptor Ligand characteristics Receptor characteristics Membrane Big signal molecules (peptides and proteins) Small, strongly hydrophilic molecules (amonoacides and their derivates) Intergral membrane proteins Intracellular Small hydrophobic molecules (steroids, vitamin D, retinoids, thyroidal hormones) Proteins in cytoplasm or in the nucleus Main typesod membranereceptors pathobiochemistry- receptors_1 26 I. Receptors - ion channels (ROC, ligand-gated channels)only receptors for some neurotransmitters (ion channelscontrolled by neurotransmitters) II. Receptors interacting with G-proteins (heterotrimeric) III. Receptors with its own catalytic activity a) guanylate-cyclase b) proteinkinase IV. Receptors cooperating with the non-receptor tyrosine kinases (eg. JAK) – receptorsfor somatotropin(GRH), prolactin, erythropoietin, interferons, interleukins and other cytokines. TEST pathobiochemistry- receptors_1 27 pathobiochemistry- receptors_1 28 Neurotransmitters- chemical signals, enablethe transfer of of nerve impulses between neuronsor between a neuron and the target cell synaptická štěrbina postsynaptic membrane receptor synapticvesicles (synaptosoms) voltage-controlledCa2 + channel depolarization wave Ca2+ • Neurotransmitter binds directly to the ion channel (ionotropic receptors) → electrical signal (neuron -neuron) • The neurotransmitter binds to a receptor that generates second messenger (metabotropic receptors) → chemical signal (eg. smooth muscle) I. Receptors – ion channels Receptors of the type of ion channels are present in the synapses, their ligands are neurotransmitters. Synapse General scheme pathobiochemistry- receptors_1 29 Membrane receptors for the neurotransmitters Ionotropic receptors - ligand-controlled ion channels (ROC), e.g. excitatory nicotinic acetylcholine - channel for Na+/K+, glutamate (CNS, some afferent sensory neurons) - channel for Na+/K+/Ca2+, inhibitory receptor GABA A (CNS) - channel for ClMetabotropic receptors activating G proteins, e.g. protein Gs adrenergic 1 a 2, receptor GABAB, dopamine D1, protein Gi adrenergic 2, dopamine D3, muscarinic acetylcholine M2 (also opens K + channel), protein Gq muscarinic acetylcholine M1, adrenergic 1. TEST pathobiochemistry- receptors_1 30 In central nervous system inhibitory GABA (minim. 50 % all synapses) glycine (prevails in the spinal cord) excitatory glutamate (more than10%) acetylcholine (about10 %) dopamine(about 1 %, in striatum 15 %) serotonine histamine aspartate noradrenalin (less than1 %, in hypotalamus5 %) adenosine Neuromodulation endorphinsand enkephalins, endozepines, delta-sleep-inducingpeptide etc. Neurotransmitters There are more than 30 different neurotransmitters (amino acids, biogenic amines caused their transformation, or very large peptides). Examples: In peripheral nervous system – efferent neurons excitatory acetylcholine noradrenaline – primary sensory afferent excitatory glutamate (Aβ fibers, tactile) substantion P (peptide) (C Aδ fiber nociceptive) TEST pathobiochemistry- receptors_1 31 Major neurotransmitter receptors Acetylcholine receptors Occurs at neuromuscular junctions of skeletal muscle and in almost all peripheral dendrites of efferent neurons. It consists of five subunits (2a, b, g, ε) penetrating the membrane. Acetylcholine is synthesized by the presynaptic neuron region of acetyl-CoA to choline and before release is stored in vesicles stored close to the active zone of the presynaptic membrane. Membrane also has the voltage-gated Ca2+ channels, which open when the action potential is expanded to the membrane. Increased levels of Ca2 + in the neuron endings activates the Ca + -dependent protein kinase, which phosphorylates synapsin and other proteins, thereby effecting fusion of the vesicles with the presynaptic plasma membrane and release of acetylcholine into the synaptic cleft. Acetylcholine binds the two subunits and his binding causes a conformational change and short influx of sodium ions into the cell and potassium ions out of the cell. This causes depolarization of postsynaptic membrane, and if the threshold is reached, potential-dependent channels are opened for Na + and the action potential arises. Once acetylcholine secretion ceases, its concentration in the cleft decreases and acetylcholine stops to bind to receptors. Acetylcholine is decomposed by acetylcholinesterase, which is bound to the surface of the postsynaptic membrane. TEST pathobiochemistry- receptors_1 32 Receptor Nicotine Muscarinic M1, M3 M2 Mechanism of action ion channel Gq Gi The second messenger DG + IP3 cAMP occurrence • autonomic ganglia neurons, • neuromuscularjunction, • chromaffin cells of the adrenal medulla • brain, • smooth muscle, •glandular cells • myocardium, • brain tubocurarine atropin Acetylcholine receptors Major neurotransmitter receptors TEST pathobiochemistry- receptors_1 33 cholinergic synapse One nerve impulse in the neuromuscularjunction releases approx. 300 vesicles, one containsabout 40,000molecules of acetylcholine; concentrationof acetylcholinein the synaptic cleft rises to 10 000 times. The mediator is rapidly hydrolyzed by acetylcholinesterase. acetylcholine receptors of postsynaptic membrane ACETYLCHOLINE membrane acetylcholinesterase choline acetate acetyl-CoA ATP depolarization wave Ca2+ Na+ choline acetyltransferase (axonal transport) reuptake TEST Acetylcholinestherase • The hydrolysis of acetylcholine to acetate and choline • It is a serine hydrolase pathobiochemistry- receptors_1 34 TEST Acetylcholinesteraseinhibitors • a) reversible: carbamates (physostigmine, rivastigmine, neostigmine) • b) irreversible: organophosphates (DFP, soman, sarin) N N OO NH CH3 CH3 CH3 CH3 H pathobiochemistry- receptors_1 35 Binding of toxic organophosphatesto cholinesteraseis donein two stages: reversible can be affected by reactivators) irreversible - formation of covalent bondsbetween enzyme and organophosphate fyzostigmin pathobiochemistry- receptors_1 36 e.g. at the neuromuscularjunction - Na + / K + ionophore:asymmetrical pentamer of four types of homologous subunitspenetratingthe membrane. conformationalchange ofsubunits, channelis in millisecondsmany times briefly opens and closes binding sites for localanesthetics, psychotropic phenothiazinesetc .. binding of two molecules of acetylcholine to 2 subunits 1 2 closed channel synaptic cleft cytosol  2 2 – – Na+ K+ massive influx of Na + less efflux of K + (depolarization) Nicotinic acetylcholine receptor of the nicotine type pathobiochemistry- receptors_1 37 Rhabdomyocytes Parasympathetic Sympathetic (skeletal muscles) innervated cells of target tissues postganglionic neurons of sympathetic pathways are almost always adrenergic Adrenergic receptors N NN N N N M1 Acetylcholine (cholinergic) receptors in peripheral efferent neurons N-nicotinic M-muscarinic pathobiochemistry- receptors_1 38 Ligands interacting with acetylcholine receptors of nicotine types D-tubocurarine- competitive antagonist of acetylcholine,prevents the opening of ionophore (depolarizationdoes not occur) paralysis of skeletal muscles pancuronium, vecuronium ad. - muscle relaxantsduring prolonged operations Succinylcholine- agonist binds more efficiently than acetylcholineand depolarizes. The persistent depolarizationleads to loss of electrical excitability of membrane. Short term myrelaxans. Botulotoxine– protein complex from Clostridium botulinum. Inhibits the release of acetylcholinefrom the nerve endings. Nicotine- binds to receptors in the peripheral and vegetative nervous system which controlsthe internal organs. Here causes increased activity of the digestive tract: increase of productionof saliva and digestive juices and the increase in activity of smooth muscles. Also increases the productionof sweat and may cause the contraction of pupil. pathobiochemistry- receptors_1 39 Type Principle of action Location M1 Gq Autonomic ganglia, CNS, exocrine gland cells M2 Gi heart, K+ channelsopening M3 Gq Smooth muscle M4 Gi CNS M5 Gq CNS Muscarinic cholinergic receptors The alkaloid atropin is antagonist of muscarinic receptors preventing acetylcholine binding. pathobiochemistry- receptors_1 40 Adrenergic synapse Neurotransmitter of most postganglionic sympathetic neuronsis noradrenaline. Some nerves can be also influenced by adrenaline. Depolarization wave Ca2+ Adrenergic receptors in membranes of the target cells dopamine--hydroxylase and synaptic vesicles (axonal transport) NORADRENALINE presynaptic adrenergic receptors mitochondrial monoamine oxidase extracellular COMT (catechol- O-methyltransferase) Partial reuptake Varicosities of the postganglionic sympathetic axons are analogous to the nerve terminals (string of pearls). TEST pathobiochemistry- receptors_1 41 • Dopamine is synthesized in the cytoplasm • Dopamine is transportedinto vesicles (ATP-dependent process, against concentration gradient). • Final hydroxylationof dopamine to noradrenaline occurs in vesicles. Synthesis and storage of catecholamines pathobiochemistry- receptors_1 42 Receptor 1  2 1  2 G-protein Gq Gi Gs Second messenger DG + IP3 cAMP  cAMP  Examples of location • GIT smooth muscle (sphincters) and skin blood vessels (contraction) • adrenergic and cholinergic nervous terminals (transmitter releasing inhibited) • pancreas (glandular secretion inhibited) • thrombocytes (agregation) • myocard (intensity and frequency of contractions increased) • smooth muscle in the uterus, bronchi (relaxation) • GIT smooth muscle (peristalsis) • pancreas (glandular secretion inhibited activated) Adrenergic receptors TEST pathobiochemistry- receptors_1 43 -Adrenergic receptors The typical effects of -stimulation 1 – tachycardia, inotropic effect in the myocard, 2 – bronchodilation, vasodilation in the bronchialtree, 3 – mobilization of fat stores, thermogenesis. AMP-cyclase-receptor g noradrenalin / adrenalin ATP AMP cAMP fosfodiesterases Gs H2O active proteinkinase Afosforylations inactive proteinkinase A TEST pathobiochemistry- receptors_1 44 Adrenergic receptors 2 a 1 2-receptors 1-receptors adenylatecyklase fosfolipase C PL C cAMP decrease Gi protein Gqprotein IP3 a diacylglycerol Increase in [Ca2+] Activation of PK C The typical effects of adrenergic 2-stimulation: 1-stimulation: glandular secretion inhibited vasoconstriction bronchoconstriction motility of GIT inhibited TEST pathobiochemistry- receptors_1 45 Inhibitory GABAA receptor Ligand-gated channel (ROC) for chloride anions. The interaction with g-aminomáselnou kyselinou (GABA) opens the channel. The influx of Cl– is the cause of hyperpolarization of the postsynaptic membrane and thus it´s depolarization (formation of an action potential) disabled. Cl– – – – – –– – 1 2 g2 2 1 heteropentamercontainig 3 subunits pathobiochemistry- receptors_1 46 Cl- GABA CYTOSOL Barbiturates Benzodiazepines (e.g. diazepam, flunitrazepam) Steroids (e.g. pregnanolone, allopregnanolone) Endozepine (diazepam-binding inhibitor, DBI) Agonist: muscimol (of Amanita muscaria) Another binding sites of GABA receptor TEST pathobiochemistry- receptors_1 47 More than eleven allostericmodulation sites for substancesincreasing the effect of endogenousGABA (calming down, reductionof anxiety and myorelaxation): anesthetic, ethanoland numerousdrugs such as benzodiazepinesmeprobamat and also various barbiturates. On the contrary, another ligands compete for benzodiazepine binding site or act also as the antagonistsof GABA (inverse agonists),  causing unease and anxiety (e.g. endogenouspeptidescalled endozepines). In brainstern and spinal cord, glycin has a similar function to GABA in brain. The inhibitory effect of glycinergic synapses is blocked by strychnine alkaloid, known seizure poison. Another binding sites of GABA receptor pathobiochemistry- receptors_1 48 Inhibitory GABAergic synapse g-Aminobutyric acid(GABA) is the main inhibitory neurotransmitter in CNS. Gabaergic synapses represent about 60 % of all brain synapses. GABA mitochondrial Synthesis of GABA from glutamate Depolarizationwave Ca2+ GABA/benzodiazepine receptors GABA id captured by glial cells, degraded to sukcinate and transformed to glutamate a glutamine Parial reuptake (transporters GAT 1-4) pathobiochemistry- receptors_1 49 Receptors of the most important neurotransmitters Receptors associated with G-proteinsIon channels (ROC) Gs (increasing cAMP) Gi (decreasing cAMP)Gq (increasing IP3 /DG) – adrenergic β1,β2,β3 adrenergic α2 adrenergic α1 Na+/Ca2+/K+ – glutamate ionophores glutamate mGluR skupiny II a III glutamate mGluR class I dopamine D1,5 dopamine D3,4 dopamine D2 – serotonin 5-HT3 serotonin 5-HT4,6 serotonin 5-HT1 serotonin 5-HT2 histamine H2 histamine H3,4 histamine H1 – – tachykinin NK1 for substance P Cl– – GABAA – glycine GABAB (metabotropic) – – – Na+/K+ – acetylcholine nicotinic acetylcholine muscarinic M2,4 acetylcholine muscarinic M1,3,5 – – – – TEST pathobiochemistry- receptors_1 50 Common structural features: All of them have seven hydrophobichelical domains,penetratethe mambrane and connect extra- and intracellular loops. H2N -COOH II. Receptors interacting with heterotrimeric G-proteins Binding site for agonist(bindingsites for antagonists are also present) Intracellulardomains bimding site for interaction with G-protein of single specific type. Few minutes Neurotransmitters Hormones Agonist-ligand causes signale transduction Antagonist- prevents pathobiochemistry- receptors_1 51 Heterotrimeric G-proteins Proteins binding GDP or GTP Mostly freely bound to cytoplasmatic membrane – they can move along its inner surface. Subunits ,  a g. Identified more than 20 types of different G subunites. Subunits G and Gg are hydrophobic and are not specific. Subunits G are the biggest, bind GDP or GTP and are specific for every type of transduction mechanism. TEST Heterotrimeric G-proteins activation cycle by interaction with receptor-specific ligand complex pathobiochemistry- receptors_1 52 receptor-specific ligand complex BIOLOGICAL EFFECT CONCENTRATION CHANGE OF„SECOND MESSENGER“ Trimer G-GDP,g receptor-ligand -trimer G-GDPg complex Dimer g GTP GDP Activated G-GTP subunit Interaction with target proteinPi Inactive G-GDP subunit Dimer g pathobiochemistry- receptors_1 53 Chosen types of G-proteins Type of G subunit Examples of activatingreceptor Effect of activatedG To target protein (intracellularsignal) Gs (stimulating) glucagon parathyrine -adrenergic Adenylatecyclase stimulation (cAMP, Ca2+) Gi (inhibitory) somatostatin 2-adrenergic Adenylatecyclaseinhibition (cAMP, K+) Gq (activating PI cascade) vasopressin V1 endothelinETA,B acetylcholineM1 1-adrenergic phospholipaseC stimulation (DG+IP3, Ca2+) Gt (inhibitory) (transducin) rhodopsin phosphodiesterasecleaving cGMP stimulation TEST Receptors activatingGs and Gi stimulate or inhibitadenylatecyclase pathobiochemistry- receptors_1 54 Adenylatecyclase - membrane enzyme catalysing reaction ATP  cAMP + PPi ; cAMP is second messanger. AMP-cyclasereceptor receptor GS g Gi g ligand ligand ATP H2O cAMP proteinkinase A inactive (R2C2) activeproteinkinase A 2 C + 2 R(cAMP)2 AMP Fosfodiesterase* Protein phosphorylation (Thr, Ser) x * Inhibition by e.g. caffeine, theophylline (METHYLXANTHINES) TEST pathobiochemistry- receptors_1 55 Effects of cAMP in cells C C R R Proteinkinase A (inactive) R R C C Protein Protein Protein-P Protein-P ADP ADP ATP ATP Proteinkinase A (active) cAMP Protein phosphorylation. In cytoplasm- most often metabolic enzymes (fast response) In nucleus–gene specific transcriptionfactor CREB (cAMP response element-bindingprotein)phosphorylation (pomalejší odpověď) AKAP AKAP pathobiochemistry- receptors_1 56 cAMP provides many different effects in the cell. One of the most important effect is proteinkinase A activation, which phosphorylates many others metabolic enzymes. The effects of kinases can by aimed for certain proteins phosphorylation. That is maintained by specific proteins binding kinases. In the case of proteinkinase A, it is about so called AKAPs (A kinase anchoring proteins), which serve as supporting structure and localize proteinkinase A position near by certain substrate, which is supposed to be phosphorylated and at the same time, their spontaneous aktivity is reduced. Proteinkinase A is heterotetrameric molecule, containing two regulatory and two catalytic subunits. In inactive state, subunits are bound to each other. cAMP binds regulatory subunits causing their separation from catalytic subunits, which become active and catalyze phoshate transmission from ATP to serine or threonine residues of target proteins. Proteinkinase A catalytic subunit also enters the nucleus where prosphorylate gen specific transcription factors so called CREB (cyclic AMP response element-binding protein). CREB binds cAMP-responsive element in unphosphorylated state and is poor transcription activator. After phosphorylation by proteinkinase A, CREB bind coactivator CBP (CREB-binding protein) causing transcription amplification. Some bacterial toxins modify G-proteins effect. Cholera is an infectious intestinal disease causing severe life threatening diarrheas. Diarrhea is caused by enterotoxin produced by bacteria Vibrio cholera. Choleratoxin is protein causing by its effect inhibition of GTPasa activity of Gs protein subunit. Modificated s subunit is „frozen“ in active state continually producing cAMP. cAMP effect is active channel for Cl− in intestinal cell membrane and its effect causes chloride ions and water secernation to intestinal lumen. Inhibitory G-protein is the target of pertusis toxin effect, which is produced with whooping cough by bacteria Bordetella pertusis. The result is Gi protein inactivation and cAMP overproduction. Besides proteinkinase A activation and proteins phosphorylation, cAMP or cGMP can bind also ion channels influencing their permeability. Those mechanisms find its use especially in activation of olfactory and visual perceptions. pathobiochemistry- receptors_1 Examples of hormones effecting through PAK activation Hormone Localization of the effect CRH Adenohypophysis TSH Thyroid follicle LH Testicular Leydig cells, yellow body (corpus luteum) FSH Ovaria follicle cells, testicular Sertolicells ACTH Adrenal cortex ADH Kidney distale tubule cells PGI2 Thrombocytes Adrenaline, noradrenaline  - receptorsin many cells glukagon Livers 57 pathobiochemistry- receptors_1 Oba produkty jsou sekundární „poslové“: Inositol-1,4,5-trisfosfát otevírá kanál pro Ca2+ v membráně ER, diacylglycerol aktivuje membránovou proteinkinázu C. III. Receptors activating Gq protein stimulate phospholipase C triggering phosphatidylinositol cascade Fosfolipáza C That kind of receptors binds q-isoform subunit. Ligand binding results in activation of enzyme phospholipase C. Activated form of this enzyme hydrolyzes membrane bound phosphatidylinositolbisphosphate (PIP2) forming two different second messangers: diacylglycerol (DG) and 1,4,5inositoltrisphosphate (IP3). IP3 has its binding site on sarko- and endoplasmatic reticulum, where stimulates Ca2+ releasing. Ca2+ ions activate enzymes containing calcium-calmodulin subunit including proteinkinase. Calcium dependent calmodulin kinase II also influence CREB in nucleus. DG localized in membrane activates proteinkinase C amplyfying response by target proteins phosphorylation. 58 pathobiochemistry- receptors_1 Both products are second „messengers“: Inositol-1,4,5-trisphosphate opens Ca2+ channel in ER membrane, diacylglycerol activate membrane proteinkinase C. III. Receptors activating Gq protein stimulate phospholipase C triggering phosphatidylinositol cascade Phospholipase C 59 pathobiochemistry- receptors_1 phospholipase Creceptor Gq g specific ligand PIP2 DG proteinkinase C activation phosphorylation [Ca2+] increasing in cytoplasm Endoplasmatic reticulum Ca2+ IP3-receptor in ER membrane ligand controlled Ca2+ ion channel IP3 Phosphatidylinositol cascade active proteinkinase C Ca2+ TEST 60 pathobiochemistry- receptors_1 Regulation of metabolism by cytoplasmatic Ca2+ concentration changes •Basal Ca2+ concentration in cytoplasm  1.10-7 mol/l •Concentration increasing to  1.10-6 fast and effectively activates different cellular process •Ca2+ increasing may be caused by Ca2+ influx through cytoplasmatic membrane (e.g. Smooth muscle contraction) releasing from intracellular supplies (ER, mitochondrie) e.g. IP3 dependent Ca2+ channel in ER or ryanodine channels in skeletal and cardiac muscle 61 pathobiochemistry- receptors_1 Regulatory protein calmodulin Increasing Ca2+ level activates numerous Ca2+-dependent proteins forming family of small calcium dependent proteins. The most important is calmodulin which is present in almost all cells. Ca2+ binding to calmodulin (4 binding sites) changes its conformation and activates its interaction with another proteins, e.g. kinases, phosphatases and others. Some of Ca-calmodulin-dependent kinases are highly specific, othershave wide substrate specificity. 62 pathobiochemistry- receptors_1 Examples of hormones effecting through phosphatidylinositol system activation and PKC Hormone Localization of the effect TRH Adenohypophysis GnRH Adenohypophysis TSH Thyroid follicle Angiotensin II/III Adrenal cortex (zona glomerulosa) Adrenaline 1- receptors 63 pathobiochemistry- receptors_1 III. A) Receptors with guanylatecyclase activity After ligand binding transform GTP to cGMP cGMP is second messenger Activates proteinkinase G Two kinds of receptors: •membrane •cytoplasmatic III. Receptors with enzymatic activity cGMP can be also second messenger. Unlike adenylatecyclase, guanylatecyclase isn´t activated by G-proteins. There are two different types of guanylatecyclase: membrane bound enzymes activated dirrectly by extracellular ligands and soluble enzymes in cytoplasm, reacting to small diffusible molecules. Both types of quanylatecyclase are located in vascular smooth muscle cells. 64 pathobiochemistry- receptors_1 Membrane receptors with guanylatecyclase activity ANP GTP cGMP + PPi proteinkinase G inactive active proteinkinaseG (PKG) Protein phosphorylation Receptorsfor ANP Present especially in vascular smooth muscle and in kidneys ANP is secernated by myocytes atria as a response to increasing of bloodvolume or pressure GMP phosphodiesterase H2O Membrane bound enzyme is receptor for natriuretic peptides (ANP, BNP, urodilatin). Receptor contains extracellular domain for ligand binding, simple transmembrane helix and intracellular guanylatecyclase domain. Guanylatecyclase activity is initiated by ANP binding to extracellular domain. Likewise cAMP and cGMP has effect through proteinkinase activation. This kinase is called proteinkinase G according the convention. Natriuretic peptides receptors are localized in vascular smooth muscle, kidneys and other tissues. ANP is secernated by myocytes atria as a response to increasing blood volume or pressure in right atrium causing vasculature relaxation. That leads to decrasing of total peripheral resistance and improving of local blood flow. In kidneys, causes dilatation of afferent and narrowing of efferent glomerular arteriole and relaxation of mesangial cells. Glomerular capillary pressure and glomerular filtration are increased and that leads to increased sodium and water excretion. TEST 65 pathobiochemistry- receptors_1 66 Cytoplasmic receptors with a guanylatecyclase activity NH2 NH2 hem NO GTP cGMP The receptor is dimeric and a binds hem NO is boundto the hem, its bond rises a catalytic activity of guanylatecyclase NO is generated by nitroxide synthase (NOS) NO goes through by membranes easily, it can be generated also by other cells and to the target cell penetratesby diffusion. Activation of proteincinase G phosphodiesterase GMP Soluble guanylatecyclase Soluble guanylatecyclase is in the cytoplasm of many cells. It is a dimeric molecule with a hem. It binds NO which causes in its structure conformation changes and rises its enzymatic activity. NO is synthetised by nitroxide synthase (NOS) from arginine. It can be also generated in organism from some exogenous compounds (NO donors), for example nitroglycerine, nitropruside. cGMP is degradeted by a few types soluble or in membrane bound cGMP phosphodiesterases. Inhibitors of cGMP phosphodiesterases cause also an increase of cGMP and prolong relaxation of smooth muscles. pathobiochemistry- receptors_1 67 Proteinkinase G cGMP sensitive proteinkinase G It is spread in a lot of tissues It phosphorylates different proteins (enzymes, transport proteins and so on) Effect of PKG in smooth muscles Phosphorylationof proteins: • inactivation of proteinswhich promotereleasing of Ca2+ from ER   Ca2+ •activation MLC phosphatase  inhibition of an actin-myosin interaction •Decrease of an activity of K+-channelswhich promotehyperpolarization  decrease of an influx Ca2+ into the cell Relaxation of smooth muscles pathobiochemistry- receptors_1 68 Meaning of NO/cGMP signalization in smooth muscles of vessels cGMP is a crucial second messenger for an induction of relaxation of smooth muscles in vessels  vasodilatation and increase flow of blood NO is produced in endotel cells by nitroxide synthase from arginine (activation for example by acetylcholine) and diffuses into adjacent cells of smooth muscle L-Arg ·NO + L-citrulline NO-synthase pathobiochemistry- receptors_1 69 R-O-NO2 nitrite ·NO Drugs like organic nitrates are a source of exogenous NO Glyceryl trinitrate Isosorbide dinitrate Therapy of angina pectoris Activation of soluble guanylatecyclase Vasodilatationeffect to arteries releases coronaryspasm and normalise a perfusion pathobiochemistry- receptors_1 70 Inhibition of phosphodiesterase potentiates the effect of NO cGMP GMP phosphodiesterase H2O Drug sildenafil (Viagra) is selective inhibitor of phosphodiesterase5 (PDE5) which is highly expressed in smooth muscles of vessels. Viagra promotes the effect of NO· which is released during sexual stimulation by inhibition of PDE5 and rises a concentrationof cGMP in corpora cavernosa. The result is a relaxation of smooth muscle in vessels and perfusion of corpora cavernosa. There are more types of phosphodiesterases, dependingin a type of cells. pathobiochemistry- receptors_1 71 III. B) Receptors with tyrosinkinase activity Collective features •binding of the signal molecule to the receptor causes conformation changes •tyrosinkinase activity of receptor is activated • it caused autophosphorylation of tyrosine of receptor alternatively other proteins (IRS) •other proteins (adapter molecules) bind to the phosphorylated receptor and substrates phosphorylated by the receptor • adapter proteins bind to phosphotyrosine residue by SH2 domains (Src homologs of 2 domains). • adapter proteins react with other molecules and a signal is carried by a cascade of phosphorylation/dephosphorylation reactions by changing of guanine nucleotides, conformation changes and so on. pathobiochemistry- receptors_1 72 Membrane receptor family with a tyrosinkinase activity is formed by receptors for growth factors and insulin. Growth factors stimulate mitosis, cell differentiation, migration of cells and apoptosis. Insulin stimulate an utilization of nutrients. Collective feature of receptors is intracellular tyrosinkinase domain. For example IGF-1 (insulin-like growth factor-1) receptor; EGF (epidermal growth factor) receptor; PDGF (platelet-derived growth factor) receptor belong into the subfamily with a tyrosinkinase activity. pathobiochemistry- receptors_1 73   -S-S- -S-S-   -S-S- Dimericstructure Binding site for insulin on - subunits Tyrosinkinase activity on -subunits Insulin receptor Insulin binding to the receptor  tyrosinkinase activity autophosphorylationof -subunitsand phosphorylationof proteins IRS 1-4 (insulin receptor substrates1-4)   -S-S- -S-S-   -S-S- Insulin -P-P P-P- IRS1-4 IRS1-4 -P PI-3-kinase binding to the membrane activation of phosphoproteinphosphatase-1 activation Ras – expression of genes Binding insulin to the receptor causes internalization of complex hormon receptor, receptors are partially recyclated TEST pathobiochemistry- receptors_1 74 Insulin receptor Insulin receptor is in membranes like a dimmer. Each monomer consist of extracellular subunit α and integral membrane subunit β. Subunits α a β are connected by disulfide bond and disulfide bond is also between monomers. Binding sites for insulin are on α subunits. Subunits β include domains with own tyrosinkinase activity. After bindind insulin β subunits phosphorylate themselves. pathobiochemistry- receptors_1 Some of the signaling pathways of insulin http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7 75 pathobiochemistry- receptors_1 The substrates of the insulin receptorIRS1-4 are adapter proteins. If the insulin-receptorcomplex phosphorylatedthese proteins, they follow more proteinsand activate them so. Glycogen synthesis Phosphorylation of IRS activates the regulatory subunit of PI 3-kinase The catalytic subunit of PI 3-kinase phosphorylates PIP2 to PIP3 PIP3 activates protein kinase B (AKT) activation of PKB (ACP) helps PDK Activated Akt diffuses into the cytoplasm and foforylates (inactivates) glykogensynthasa kinase Glycogen synthesis is activated (the active form of glycogen synthase is dephosphorylated) Translocation of glucose transporters Insulin receptor phosphorylates CBI (IRS) CBI-CAP complex translocates into lipid raft membrane CBI is reacted with an adapter proteinCrk Crk associated with C3G C3G activates TC10 (G-protein) Activates the translocation of transporters into the plasma membrane Examples mechanism of action of insulin receptor 76 pathobiochemistry- receptors_1 The receptor for epidermal growth factor EGF Once ligand binding occurs receptor dimerization R R R R -P -PPP- SoS Ras–GTP Raf phosphorylation cascadeMAP phosphorylation This activates the tyrosine kinase activity in the cytoplasmaticdomain. Receptor autophosphorylation The phosphorylatedsites bind Grb2 adapterprotein (SH-2 domains). Through SOS protein is activated by G-protein Ras  aktivace MAPkinase cascade (Ras / MAP-cascade) Receptor with tyrosine kinase activity 77 pathobiochemistry- receptors_1 Ras is a monomeric G-protein (structural analogue  -subunits) SoS Activation of Ras - a key step in signal transduction. Inactive Ras-GDP switches to active Ras -GTP, which activates anothermember of the pathway. Inactivation of Ras - subsequenthydrolysis of GTP by using activating protein GTPase activity of G-protein Monomer G-protein - binds GTP and has simultaneously GTPase activity. Activated GTP binding site GDP 78 pathobiochemistry- receptors_1 Ras superfamily proteins 5 families: Ras, Rho, Arf, Rab, Ran Anchored to the lipid membrane by lipid anchors (myristoyl, farnesyl) Monomer G-proteins, which play an important role in regulating growth, morphogenesis, cell motility, cytokinesis like. Mutations in the Ras genes induce proliferation and pathologic antiapoptosis. Ras Mutations occur in about 30% of all human cancers. 79 pathobiochemistry- receptors_1 MAP-kinase signaling pathway (Mitogen activated protein kinase) Map-kinase cascade Ras–GTP MAP-kinase-kinase-kinase MAP-kinase-kinase MAP-kinase Phosphorylation of membrane proteins or cytosolic Phosphorylationof regulatory proteinsin the nucleus, promotion of proliferation (e.g. activation of transcriptionfactors Jun, Fos) ATP ATP ATP ADP P ADP ADP ADP ATP P P MAPKKK, Raf Especially regulates cell growth and differentiation. MEK ERK Described 3 systems, the most famous ERK. 80 pathobiochemistry- receptors_1 Abbreviation Name Function PDGF Derived Growth Factor platelet mitogen for connective tissue cells and undifferentiated neuroglia EGF Epidermal growth factor mitogen series of cells of mesodermal origin and ectodermal FGF-2 Fibroblast growth factor 2 mitogen for a variety of cells such as fibroblasts, endothelial cells, myoblasts; induces embryonic mesoderm IL-2 Interleukin 2 mitogen for T-lymphocytes The mitogens - growth factors supporting the proliferation Examples of mitogen: 81 pathobiochemistry- receptors_1 Receptors with a serine / threonine kinase activity The ligand is e.g. transforming growth factor-  (TGF- ) After ligand binding receptorsdimerize and activate the serine / threoninekinase activity in their respective domains One active subunit phosphorylatespartner subunit in the catalytic site Phosphorylated partner subunit phosphorylatesthe cytoplasmic proteinsSMAD Smad proteins are activated by phosphorylation and forms dimers with other SMAD proteins The translocationto the nucleus, where they interact with other regulation proteins P P P P Migration to nucleus SMAD SMAD 82 pathobiochemistry- receptors_1 IV. Receptors activating non-receptor tyrosine kinase ligand dimerization –PSTAT STATSTAT JAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription) Receptorhas kinase activity, but is associated with the tyrosine kinase JAK. After ligand binding receptorsdimerize (homodimers or heterodimers) Activated somehow phosphorylatetyrosine residues on the receptor. The sites phosphorylatedadapterproteinsbind STAT (using SH2 domains) STAT are phosphorylatedand dimerize. STAT dimers translocateto the nucleus, where they act as transcriptionfactors –P STAT –PSTAT TEST 83 pathobiochemistry- receptors_1 JAK-STAT receptors - a family of receptors for cytokines * (e.g.interferons, interleukins) Diverse cytokine effects are caused by the existence of large amounts of STAT proteins Receptorsfor various cytokines bind the various states which producesheterodimersin different combinations Thus, it is possible that different cytokines affect different genes Receptorscooperatingwith the JAK-STAT also have prolactin, erythropoietinad. * Cytokines - small signaling proteins involved in the immune response significantly. They are produced by immune cells (macrophages, T-cells etc.) and are capable of inducing such as rapid division and differentiation of certain cell types involved in the fight against pathogens and other features of the immune defense 84 pathobiochemistry- receptors_1 Control Membrane Receptors Regulation by changing the number of receptors(down regulation, up- regulation) Controlproperties receptor (desensitization) For example: desensitization of -adrenergic receptor Upon binding of the ligand to the receptoractivates BARK (β-adrenergic receptor kinase) The cytoplasmic portion of the receptormolecule, the phosphorylation The phosphorylatedsite binding arrestin protein that inhibits the ability to activate G-protein 85 pathobiochemistry- receptors_1 Intracellular receptors Steroid hormones, calcitriol, and iodo thyronine Retinome Receptors are located in the cytoplasm or in the nucleus The hormone-receptor complexes bind to specific DNA sites and serve as transcription factors Hormone-receptor complex to the DNA binding site HRE (hormone response element) Superfamily of steroid and thyroid receptors - a family of structurally related proteins. Activation of transcription is a slower process, a response delay TEST 86 pathobiochemistry- receptors_1 Example cortisol (glucocorticoid GK, their receptors GR) hydrophobicmoleculepenetratesthe cell hormone inactive receptor glucocorticoid (GR) exists in cytoplasm as a complex with a protein dimer hsp 90 (chaperone) and other proteins active monomeric complex cortisol-receptor, separated hsp 90 proteins with others active complex and dimerizes nukleoporus translocated into the nucleus GR kortisol CBG cortisol in the extracellularspace transmitted CBG (corticosteroid-bindingglobulin) 87 pathobiochemistry- receptors_1 Dimeric complex cortisol receptorin the nucleus continues to site-specific dsDNAsequences of GRE (glucocorticoidresponse element), i.e. on the HRE (hormoneresponse element) specific to glucocorticoids. DNA binding domain GR receptor DNA GRE Binding domain for cortisol (hydrophobic pocket) 88 pathobiochemistry- receptors_1 GR dimer – intracellular glucocorticoid receptor (dimer) GRE – glucocorticoid response element GREB protein – GRE binding protein (specific transcription factor) TF IID Pol II CTD > 1 000 bp Mediator proteins enhancer koactivatorGREB protein GRE dimeric complex cortisol-GR promotor basal Transcriptionalapparatus Active complex cortisol receptor binds to DNA in a sequence-specific site GRE (glucocorticoid responseelement, one of the many HRE - hormoneresponse elements). Complex itself, however, hormone-receptor binding to DNAand affect transcriptionis not capable. Connectsto a specific coactivatorproteins GREB(glucocorticoid response element-bindingproteins). This complex via mediatorproteinsbuilds upon the basal transcriptional apparatusto the promoter and initiates transcription. Initiation of transcription cortisol 89 pathobiochemistry- receptors_1 Overviewof the most importantneurotransmitterreceptors Cholinergic synapsis Nicotine MuscarinicReceptor Mechanism of action Second messenger Location Block receptor Ion channel Tubokurarine Atropine •Brain •Smooth muscle •Glandular cells •Myocardium •Brain •autonomic ganglia neurons •neuromuscular junction •chromaffin cells of the adrenal medulla DG + IP3 cAMP M1, M3, M5 M2, M4 Gq Gi TEST 90 pathobiochemistry- receptors_1 Adrenergic synapses Second messenger Examples of location •smooth muscles of the GIT (sphincters) and skin blood vessels (contraction) •adrenergic and cholinergic nerve endings (inhibition of transmitter release) •pancreas (exocrine secretion inhibition) •platelets (aggregation) •myocardium (increase in strength and frequency of contractions) •smooth muscle of the uterus, bronchus (relaxation) •smooth muscle of GIT (peristalsis) •pancreas (exocrine secretion activation) The actionof the neurotransmitters in the autonomic nervous system System Parasympaticus Sympaticus Mediator Acetylcholine Acetylcholine Noradrenaline Receptor Location nicotine muscarinenicotine muscarine •dendrites ganglion neurons •membrane s of target cells •sweat glands •membranes of target cells •chromafinní buňky dřeně nadledvin •dendrity neuronů ganglií TEST 91