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Chaps 4 & 5: Receptors as drug targets, structure, function & signal transduction Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. Communication allows cells to perceive and correctly respond to their microenvironment for proper development, tissue repair, and immunity and normal tissue function. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, diabetes and more. Understanding cell signaling is crucial for treating diseases effectively. neighbor g talk self lf talk cell 1 signals signals ((neurotransmitters)) receptors cell 2 cell 1 cell 2 synapse cell 2 receptor a signals (hormones) signals i l (hormones) cell 1 cell 5 receptor c signals (hormones) signals (hormones) cell 3 synapse receptor b The same signal can deliver different messages to different tissues (histamine). (histamine) etc. synapse cell 3 cell 4 receptor d Important functions of receptors: 1. Globular proteins (receptors) acting as a cell’s ‘letter boxes’ 2 Located mostly in the cell membrane 2. 3. Receive messages from chemical messengers coming from other cells 4. Transmit a message into the cell leading to a cellular effect 5. Different receptors specific for different chemical messengers 6. Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers © Oxford University Press, 2013 1 Signals can be divided into the 5 catagories below. Signal carriers (S) have to reach the proper receptors (R) for the message to be recieved. Intracrine signals are produced by the target cell that stay within the target cell. cell a S R S = signal R = receptor Autocrine signals are produced by the target cell, are secreted, and affect the target cell itself via receptors. Sometimes i autocrine i cells ll can target cells ll close l b if they by h are the h same type off cell ll as the h emitting cell. An example of this are immune cells. cell a S R Juxtacrine signals target adjacent (touching) cells. These signals are transmitted along cell membranes via protein or lipid components integral to the membrane and are capable of affecting either the emitting cell or cells immediately adjacent. R cell ll a S R cell ll b Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example. cell ll a S R cell b S synapse R cell c S synapse etc. synapse Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to reach all parts of the body (long-range allostery). S R2 cell c S S S S S S S cell a S S R3 cell d S S S R1 cell b © Oxford University Press, 2013 2 Central Nervous System (CNS) Brain and spinal cord Integrative and control centers, process info out and info in structure function Peripheral Nervous System (PNS) Cranial nerves and spinal nerves Communication lines between the CNS and the rest of the body Sensory (afferent) division Motor (efferent) division (CNS) Somatic and visceral sensory nerve fibers Conducts impulses from receptors to the CNS Motor nerve fibers Sympathatic division mobilizes body systems during activity (fight or flight) Parasympathetic division Conducts impulses from the CNS to effectors (muscles and glands) Autonomic nervous system (ANS) Somatic nervous system Visceral motor (involuntary) Somatic motor (voluntary) Conducts impulses from the CNS to cardiac muscles, smooth muscles, and glands Conducts impulses from the CNS to skeletal muscles Conserves energy C Promotes 'housekeeping' functions during rest © Oxford University Press, 2013 3 Central nervous system The central nervous system consists of the two major structures: the brain and spinal cord. The brain is encased in the skull, k ll andd protected t t d by b the th cranium. i The spinal cord is continuous with the brain and lies on the backside to the b i andd is brain, i protected t t d by b the th vertebra. t b The spinal cord reaches from the base of the skull, continues through or starting below the foramen magnum, and t terminates i t roughly hl level l l with ith the th first fi t or second lumbar vertebra, occupying the upper sections of the vertebral canal. The CNS iintegrates Th t t iinformation f ti it receives from, and coordinates and influences the activity of, all parts of the body and it contains the majority of the nervous system. t Usually, U ll the th retina ti andd the optic nerve (2nd cranial nerve), as well as the olfactory nerves (1st) and olfactory epithelium are considered as parts of the CNS CNS, synapsing s napsing directl directly on brain tissue without intermediate ganglia.. © Oxford University Press, 2013 4 Peripheral nervous system The peripheral nervous system (PNS) is the part of the nervous system that consists of the nerves and ganglia on the outside of the brain and spinal cord. cord Its main function is to connect the central nervous system (CNS) to the limbs and organs, essentially serving as a communication relay going back and forth between the brain and spinal cord with the rest of the body. Unlike the CNS, the PNS is not protected by the bone of spine and skull, or by the blood–brain barrier, which leaves it exposed to toxins and mechanical injuries. The peripheral nervous system is divided into the somatic nervous system and the autonomic nervous system 5 © Oxford University Press, 2013 The autonomic nervous system is responsible for regulating the body's unconscious actions. The parasympathetic system is responsible for stimulation of "rest-and-digest" or "feed and breed" activities that occur when the body is at rest, especially after eating, including sexual arousal, salivation,, lacrimation ((tears), ), urination,, digestion g and defecation. Its action is described as being g complementary to that of the sympathetic nervous system, which is responsible for stimulating activities associated with the fight-or-flight response. Parasympathetic nervous system Sympathetic nervous system © Oxford University Press, 2013 6 Somatic nervous system y The somatic nervous system is the part of the peripheral nervous system associated with skeletal muscle voluntary control of body movements. It consists of afferent nerves (toward) and efferent nerves (out of). of) Afferent nerves are responsible for relaying sensation from the body to the central nervous system (CNS); efferent nerves are responsible for sending out commands from the CNS to the body. There are 43 segments of nerves in the human body. With each segment, there is a pair of sensory and motor nerves. In the body, 31 segments of nerves are in the spinal cord and 12 are in the brain stem. stem The somatic nervous system consists of three parts: Spinal nerves: They are peripheral nerves that carry sensory information i f i into i andd motor commands d out off the h spinal cord. Cranial nerves: They are the nerve fibers that carry information into and out of the brain stem. They y include smell, vision, eye, eye muscles, mouth, taste, ear, neck, shoulders, and tongue. Interneurons (association nerves): These nerves integrate sensory input and motor output, output numbering thousands © Oxford University Press, 2013 7 Nerve cells make contact to muscle cells, organs and other nerve cells Can be 1000s of axon inputs p from other nerve cells. … or synaptic button when contact to neuron 8 © Oxford University Press, 2013 axon Synaptic button or neuromuscular junction Cell body © Oxford University Press, 2013 9 Nerve cells and glial cells are incredibly complicated networks of connections. Perhaps, 100,000,000,000 neurons with 10,000 connections each 1,000,000,000,000,000 (quadrillion). 10 © Oxford University Press, 2013 Neurological disorders Charcot–Marie–Tooth disease (CMT), also known as hereditary motor and sensory neuropathy (HMSN) hhereditary (HMSN), dit sensorimotor i t neuropathy th andd peroneall muscular l atrophy, t h is i a heterogeneous h t inherited disorder of nerves (neuropathy) that is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. Presently incurable, this disease is one of the most common inherited neurological l i l disorders, di d with ith 37 in i 100,000 100 000 affected ff t d (1 ( 1 in i 2500). 2500) Alzheimer's disease (AD), is a neurodegenerative disease characterized by progressive cognitive deterioration together with declining activities of daily living and neuropsychiatric symptoms or b h i l changes. behavioral h Th The di disease process iis associated i t d with ith plaques l andd tangles t l in i the th brain. b i The Th most striking early symptom is loss of short-term memory (amnesia), which usually manifests as minor forgetfulness that becomes steadily more pronounced with illness progression, with relative preservation of older memories. As the disorder progresses, cognitive (intellectual) impairment extends t d to t the th domains d i off language, l skilled kill d movements t andd recognition, iti andd functions f ti suchh as decision-making and planning become impaired. Parkinson's disease (PD), is a degenerative disorder of the central nervous system that often i impairs i the th sufferer's ff ' motor t skills kill and d speech. h Parkinson's P ki ' di disease belongs b l to t a group off conditions called movement disorders. It is characterized by muscle rigidity, tremor, a slowing of physical movement, and in extreme cases, a loss of physical movement). The primary symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused b the insufficient by ins fficient formation and action of dopamine, dopamine which hich is produced prod ced in the dopaminergic neurons of the brain. Secondary symptoms may include high level cognitive dysfunction and subtle language problems. PD is both chronic and progressive. © Oxford University Press, 2013 11 Myasthenia gravis is a neuromuscular disease leading to fluctuating muscle weakness and fatigability during simple activities. Weakness is typically caused by circulating antibodies that block acetylcholine receptors at the post-synaptic neuromuscular junction, inhibiting the stimulative effect of the neurotransmitter acetylcholine. Demyelination results in the loss of the myelin sheath insulating the nerves. When myelin degrades, conduction of signals along the nerve can be impaired or lost, and the nerve eventually withers. This leads to certain neurodegenerative disorders like multiple sclerosis, Guillain-barré syndrome and chronic inflammatory demyelinating polyneuropathy. Axonal degeneration Although most injury responses include a calcium influx signaling to promote resealing of severed parts, axonal injuries initially lead to acute axonal degeneration, which is rapid separation of the proximal and distal ends within 30 minutes of injury. Early changes include accumulation of mitochondria in the paranodal regions at the site of injury. Endoplasmic reticulum degrades and mitochondria swell up and eventually disintegrate. The axon undergoes complete fragmentation. The process takes about roughly 24 hrs in the PNS, and longer in the CNS. Diabetic neuropathies are nerve damaging disorders associated with diabetes mellitus. These conditions are thought to result from diabetic microvascular injury involving small blood vessels that supply nerves (vasa nervorum) in addition to macrovascular conditions that can culminate in diabetic neuropathy. Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuropathy multiplex; diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy. Diabetic neuropathy affects all peripheral nerves including pain fibers, motor neurons and the 12 autonomic nervous system. Symptoms usually develop gradually over years. © Oxford University Press, 2013 cell potential = -50-89 mV because K+ can diffuse out, but negatively charged proteins cannot. Sodium ions cannot move in because their ion channels are closed until triggered by a signal from the presynaptic side of the neuron. Neurotransmitters are released from the prior cell and diffuse across the synapse to bind to the ion-gate receptor and open an ion gate channel (Na+ by acetyl choline, Cl -by gama aminobutyric acid and others), Na+ influx depolarizes the cell down the axon and allows Ca+2 in which causes vesicles to fuse with membrane and release the neurotransmitter neurotransmitter, starting the next impulse. neurotransmitters at synapse y p neurotransmitters bind to receptors on the next cell at the synapse Some of these are activated by the change i membrane in b potential. t ti l depolarization milli seconds proteins Axon neurotransmitters leave cell by exocytosis 0 K action potential Na Na K Na Ca+2 action potential Cl Cell Body Ion gate opens when neurotransmitter complexes at receptor. Ca+2 C K Na Cl dendrites Ca+2 H O O O O acetyl choline is an important neurotransmitter, Ca+2 B H B H N O O O O choline retaken up by presynaptic neuron H OH N diffuses across the synaptic cleft and binds to a Na+ ion-gate channel on the OH of a serine residue of the receptor H O O B B receptor H B H N H B O B H receptor receptor hydrolyzed back to the serine "OH" © Oxford University Press, 2013 13 Hormones are signaling molecules produced by glands in multicellular organisms that are transported by the circulatory system to target distant organs to regulate physiology and behaviour. Hormones have diverse chemical structures, mainly of 3 classes: icosanoids, steroids, and amino acid derivatives (amines, peptides, and proteins). The glands that secrete hormones comprise the endocrine signaling system. The term hormone is sometimes extended to include chemicals produced by cells that affect the same cell (autocrine or intracrine signalling) or nearby cells (paracrine signalling). T3 T4 14 © Oxford University Press, 2013 15 © Oxford University Press, 2013 16 © Oxford University Press, 2013 H H H H cholesterol HO many steps, plus sunshine H H H 7-Dehydrocholesterol HO H H pre-vitamin D3 H HO H H vitamin D3 HO © Oxford University Press, 2013 17 Hormone binding to receptor - insulin (Insulin protein requires post translational modification) 1. Insulin binds to tyrosine kinase-linked receptor. 2. Phosphorylation of receptor, starts sequence q leading g to synthesis of glycogen, storage of glucose (and fat too) 3. Allows influx of gglucose 4. Insulin-like molecules are even found in the simplest unicellular eukaryotes, over 1 billion yyears old 5. Our insulin is very similar to pig and cow insulin, was used before our genes were spliced p into ggenetically y modified bacteria (glucose transport) http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cfm © Oxford University Press, 2013 18 Icosanoids are signaling molecules made by oxidation of either 20-carbon omega-3 (-3) or omega6 (-6) fatty acids. In general, the -6 eicosanoids are pro-inflammatory; -3s are much less so. There are multiple subfamilies of eicosanoids, eicosanoids including the prostaglandins, prostaglandins thromboxanes, thromboxanes and leukotrienes, as well as the lipoxins and eoxins, and others. They exert complex control over many bodily systems; mainly in growth during and after physical activity inflammation or immunity after the intake of toxic compounds and pathogens, activity, pathogens and as messengers in the central nervous system. Many are classified as hormones. The networks of controls that depend upon icosanoids are among the most complex in the human body. The amounts and balance of fats in a person person'ss diet will affect the body body'ss icosanoid icosanoid-controlled controlled functions, with effects on cardiovascular disease, triglycerides, blood pressure, and arthritis. prostaglandins O thromboxanes O OH HO OH leukotrienes O O OH OH OH O OH alprostadil - inhibits blood clotting , vasodilator O thromboxane A2 - vasoconstrictor, hypertensive, yp blood clotting, g aspirin inhibits its formation lipoxin B4 - eoxin, proimfalmmatory (white blood cells), mast cells, cells contributes to allergies, allergies various cancers, Hodgkin's lymphoma © Oxford University Press, 2013 19 Steroids are organic compounds with four rings arranged in a specific configuration. Examples include the dietary lipid cholesterol and the sex hormones estradiol and testosterone. Dexamethasone is an anti-inflammatoryy drug. g Steroids have two principal biological functions: certain steroids (such as cholesterol) are important components of cell membranes which alter cell membrane fluidity, and many steroids are signaling molecules which activate steroid hormone receptors and can lead to DNA activation and protein synthesis. synthesis C A D B glucocorticoids (adrenal cortex) mineralocorticoids (adrenal cortex) estrogens (gonads) androgens (gonads) progestins (ovaries and placenta) vitamin D (diet and sun) ...and more Letters are used to identify the 4 rings. 20 © Oxford University Press, 2013 CH3 OH H H H H HO Estradiol is the primary female sex hormone. It is important in the regulation of the estrous and menstrual female reproductive cycles. Estradiol is essential for the development and maintenance of female reproductive tissues but it also has important effects in many other tissues including bone. While estrogen l l in levels i men are lower l compared d to t women, estrogens t h have essential ti l functions f ti i men as well. in ll It is i 2 steps t away from progesterone. CH3 OH H H CH3 H H O Testosterone is secreted primarily by the testicles of males and, to a lesser extent, the ovaries of females. Small amounts are also secreted by the adrenal glands. In men, testosterone plays a key role in the d l development off male l reproductive d i tissues i such h as the h testis i and d prostate as well ll as promoting i secondary d sexual characteristics such as increased muscle, bone mass, and the growth of body hair. Levels of testosterone in adult men are about 7–8 times as great as in adult females. As the metabolic consumption of testosterone in males is greater, the daily production is about 20 times greater in men than women. 21 © Oxford University Press, 2013 H Common steroid transformations while similar in appearance, everyone of these steroids has a specific signal to transmit, such as turning on a genetic switch. They are extremely powerful chemicals, some operating at the nanomolar level. H3C V22, p. 703, p 1263 H3C CH3 CH3 H lanosterol - the compound from which all steroids are derived. CH3 O HO 3 sp oxidation CH3 C H H CH3 CH3 CH3 P O O P isopentenyl phosphate dimethylallyl phosphate HO O S CoA H H 17-hydroxyprogesterone - sometimes given to prevent premature birth OH sp oxidation O CH3 H progesterone - involved in menstrual cycle and pregnacy sp3 oxidation OH O CH2 C H NADH CH3 OH H CH3 H H H H 11-deoxycorticosterone intermediate to aldosterone O O 11-deoxycortisol (cortodoxone) glucocorticoid (similar to cortosol) sp3 oxidation CH3 HO sp3 oxidation C CH2 H CH3 H H H reduced C=C is dihydrotestosterone H H (maybe with FADH2) O cortisol - glucocorticoid, increases (more potent by 3x) blood sugar, decreases immune O testosterone - produced system (hydrocortisone) in all vertebrates, 20x more in males, male sex characteristics. three well known members of the estrogen family OH CH3 OH see CH3 CH3 reactions below H H H O 5 categories of steroid hormones O 1. progestins - overies and placenta- mediate menstrual cycle and maintains pregnacy 2. glococorticoids - adrenal cortex - affect H H metabolism in diverse ways, ways decrease H OH inflamation,incease resistance to stress 3. mineralocorticoids - adrenal cortex - maintain H H H H salt and water balance H H 4. androgens - gonads - maturation and function NAD+ HO of secondary sex organs, particularly in males, sp3 oxidation HO estrone - known femaleoxidation -estradiol - prominant estrogen HO O male sexual differentiation in reproductive years, more potent 5. estrogens - gonads - maturation and function of estriol - higher levels in pregnancy, carcinogen, causes breast tenderness, than estriol (10x) and estrone (80x) in men causes anorexia secondary sex organs, particularly in females marker of fetal health CH2 C CH3 HO OH OH O OH O CH2 C CH3 H H H CH3 acetyl CoA sp3 oxidation CH3 dehydroisoandrosterone d h d i d t - most abundant b d circulating steroid, many uses CH3 O H CH3 H O H many steps H CH3 C CH3 O H H O O CH3 C OH Statins drugs work here. O NAD+ and isomerization O 3 ? steps H pregnenolone neuroactive steroid HO O 17-hydroxypregnenolone - higher levels are found at end of puberty and in pregnacy. O CH3 H HO O H OH NAD+ and isomerization H squalene H H O OH CH3 CH3 cholesterol - essential component of cell walls, precursor of steroid hormones, bile acids and vit D O squalene oxide H ? steps H about 19 steps CH3 H CH3 CH3 C CH3 H CH3 H3C O CH3 H HO H sp3 oxidation and NAD+ oxidation corticosterone - stress hormone, immune system O H O C HO OH C CH2 OH CH3 H H H aldosterone - Na+,K+ balance, blood pressure reg. © Oxford University Press, 2013 22 Amino acid derivatives (or from amino acids) – thyroxine, histamine, catacholamines (epinephrine, norepinephrine) oxytocin, cytokines, glutamate, glycine, gama aminobutyric acid, acetylcholine (neurotransmitters and hormones) O HO O O HO H2 N HO epinephrine adrenaline HO HO NH3 HO norepinephrine O NH3 glutamate I O O HO O O -aminobutyric acid glycine I O H3N H3N NH3 O I th thyroxine i (T4) HO N NH3 HO histamine Leu Pro O N N nitrosyl radical di l (nitric oxide) Cys oxytocin - 8 amino acid peptide I O NH3 HN dopamine Gly O S O acetylcholine Tyr S Cys Asn Ile © Oxford University Press, 2013 23 Chemical Messengers (deliver messages), Receptors (receive messages) Neurotransmitter: A chemical released from the end of a neuron which travels across a synapse to bind with a receptor p on a target g cell,, such as a muscle cell or another neuron. Usually y short lived and responsible for messages between individual neurons. Hormone: A chemical released from a cell or a gland and which travels some distance to bind with receptors p on target g cells throughout g the body y Chemical messengers ‘switch on’ receptors without undergoing a reaction (different from enzymes) Nerve 1 Synaptic button =, connections to neurons, acetylcholine l h li receptors Blood supply, supply ee.g. g insulin – absorb glucose Glucagon – release glucose Hormone Neuromuscular end plates = connections to muscles, adrenoline response Nerve 2 Cell secondary messenger Neurotransmitters © Oxford University Press, 2013 24 Structure and function of receptors Mechanism Induced fit Messenger Messenger Messenger Cell Membrane Receptor Receptor Cell Cell Receptor Cell message Message M Receptors contain a binding site (hydrophobic hollow or cleft on the receptor protein) that is recognised by the chemical messenger Binding of the messenger involves intermolecular bonds with amino acids ion - ion H bonds dipole - dipole dispersion forces Binding results in an induced fit of the receptor protein and the messenger (alters shape) Change in receptor shape results in a ‘domino’ effect (no reaction/catalysis, different from enzyme) Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell (amplification of signal is common) Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound (could find another receptor) © Oxford University Press, 2013 25 Binding interactions • Ionic • H-bonding • van der Waals Induced fit - Binding site alters shape to maximise intermolecular bonding Phe Phe O O H Ser Ser CO2 Asp Intermolecular bonds not optimum length for maximum binding strength Induced Fit H CO2 Asp Intermolecular bond lengths optimised, change of shape causes a change in function (on or off) © Oxford University Press, 2013 26 Overall Process of Receptor/Messenger Interaction M M M = messenger R = receptor M RE R RE Signal transduction Binding Bi di interactions i i must be b strong enoughh to hold h ld the h messenger sufficiently ffi i l long l for signal transduction to take place Interactions must be weak enough to allow the messenger to depart Implies a fine balance Designing molecules with stronger binding interactions can result in drugs that block the binding site = antagonists (agonists turn on the effect) © Oxford University Press, 2013 27 Receptor Superfamilies RESPONSE TIME •ION CHANNEL RECEPTORS •G PROTEIN COUPLED RECEPTORS •G-PROTEIN •KINASE LINKED RECEPTORS msecs MEMBRANE BOUND seconds d minutes •INTRACELLULAR RECEPTORS 28 © Oxford University Press, 2013 Ion Channel Receptors General principles • Receptor protein is part of an ion channel protein complex • Receptor binds a messenger leading to an induced fit • Ion channel is opened or closed • Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+) • Ions flow across cell membrane down concentration gradient • Polarises or depolarises nerve membranes • Activates or deactivates enzyme-catalysed reactions within cell © Oxford University Press, 2013 29 Ion Channel Receptors - General principles hydrophilic tunnel Cell membrane hydrophobic exterior MESSENGER Extra cellular Extra cellular ION CHANNEL (closed) Cell membrane Cell Ion channel ION CHANNEL ( (open) ) RECEPTOR BINDING SITE Induced fit and opening of ion channel Lock Gate Ion channel MESSENGER Cell membrane Cell membrane Ion channel Ion channel Cell membrane Cell © Oxford University Press, 2013 30 Ion Channel Receptors - Gating Receptor Binding site Cell membrane Messenger Induced fit Cell membrane ‘Gating’ (ion channel opens) Five glycoprotein subunits traversing cell membrane Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl- (e.g. GABAA) = inhibitory 31 © Oxford University Press, 2013 Ion Channel Receptors Structure of protein subunits (4-TM (4 TM receptor subunits) Neurotransmitter binding region Cell membrane one of these Cell membrane Extracellular loop H2N CO2H TM1 Intracellular loop p TM2 TM3 TM4 Variable loop 4 Transmembrane (TM) regions (hydrophobic on the outside of channel) © Oxford University Press, 2013 32 Ion Channel Receptors p - Detailed Structure of Ion Channel Protein subunits TM4 TM1 TM2 TM3 TM1 TM2 TM2 TM4 TM3 TM3 TM1 TM3 TM4 TM4 TM2 TM2 TM1 TM1 TM3 TM4 Transmembrane regions Note: TM2 of each protein subunit ‘lines’ the central pore (TM2 is more polar, perhaps bonded to very polar carbohydrates, TM1, TM33 and d TM4 aree more o e hydrophobic yd op ob c and d face ce towards ow ds ce cell membrane.) e b e.) © Oxford University Press, 2013 33 Ion Channel Receptors - Gating Cell membrane TM2 Ion flow TM2 TM2 TM2 TM2 TM2 TM2 TM2 TM2 Transverse view TM2 TM2 Transverse view TM2 Closed Open • Chemical messenger binds to receptor binding site • Induced fit results in further conformational changes • TM2 segments rotate to open central pore • Fast response measured in msec • Ideal for transmission between nerves • Binding of messenger leads directly to ion flows across cell membrane • Ion flow can lead to secondary effects (signal transduction) • Ion concentration within cell alters • Leads to variation in cell chemistry © Oxford University Press, 2013 34 Ion Channel Receptors – Nicotinic receptor Binding Bi di sites Ion channel Cell membranee Two ligand binding sites mainly on -subunits subunits x subunits H3C O N H3C O CH3 H3C CH3 N CH3 H acetylcholine neurotransmitter H3C N O CH3 CH3 nicotine protonated at bodyy p p pH Muscarine OH Muscarinic agonists activate muscarinic receptors while nicotinic agonists activate nicotine receptors. Both are direct-acting cholinomimetics that mimic acetyl choline and act on the CNS. © Oxford University Press, 2013 35 Ion Channel Receptors p – Glycine y receptor p Binding sites Ion channel Cell membrane x xx subunits subunits O H3N O glycine Three ligand binding sites on -subunits The glycine receptor, or GlyR, is the receptor for the amino acid neurotransmitter glycine. GlyR is an ionotropic receptor that produces its effects through chloride current. It is one of the most widely distributed inhibitory receptors in the central nervous system and has important roles in a variety of physiological processes, especially in mediating inhibitory neurotransmission in the spinal cord and brainstem. © Oxford University Press, 2013 36 G-Protein Coupled Receptors – General principles • Receptor binds a messenger leading to an induced fit • Opens a binding site for a signal protein (G-protein) • G-Protein binds,, is destabilised then split p G protein messenger iinduced d d fit closed open G-protein split into α and β/ subunits 37 © Oxford University Press, 2013 G-Protein Coupled p Receptors p – General p principles p • G-Protein subunit activates membrane bound enzyme • Binds to allosteric binding site • Induced fit results in opening of active site • Intracellular reaction catalysed (makes cyclic AMP, PIP2, etc.) Enzyme Enzyme α α active site (closed) active site ( (open) ) IIntracellular t ll l reaction © Oxford University Press, 2013 38 G-Protein G P t i Coupled C l d Receptors R t - Structure St t Single protein with 7 transmembrane regions (7-TM receptor) Extracellular loops NH3 extracellular Membrane N -Terminal chain VII VI V IV III II Transmembrane helix I G-Protein binding region O 2C C -Terminal chain cytoplasm Variable intracellular loop Intracellular loops G protein splits into an subunit and an / subunit. 39 © Oxford University Press, 2013 G-Protein Coupled Receptors – Ligand binding site varies depending on receptor type Ligand A B C D A) Monoamines: pocket in TM helices B) Peptide hormones: top of TM helices + extracellular loops + N-terminal chain C) H Hormones: extracellular t ll l loops l + N-terminal Nt i l chain h i D) Glutamate: N-terminal chain Example receptors - ligands • Monoamines - e.g. dopamine, histamine, noradrenaline, acetylcholine • Nucleotides - e.g. Adenine, guanine, cytosine, uracil, thymine • Lipids - e.g. Cholesterol, fatty acid, triglyceride, phospholipids • Hormones - thyroxine, y , testosterone,, estrogen, g , oxytocin, y , epinephrine p p ++ • Glutamate, DOPA, glycine, acetylcholine, Ca © Oxford University Press, 2013 40 SIGNAL TRANSDUCTION neurotransmitter or hormone signal binds at receptor site of 7 TM receptor Overview There are many different types of G proteins with about 20 different types of α subunit proteins. G protein , , subunits subunit splits off and binds to another protein to start signal cascade inside the cell G protein-coupled receptors (7 TM receptors) imbedded in membranes continuing reactions inside cell protein protein various actions inside cell © Oxford University Press, 2013 41 DRUG TARGETS SIGNAL TRANSDUCTION Signal Transduction involving Gs-Proteins provide one step in signal transduction pathway. GS-Protein - membrane bound protein of 3 subunits () - S subunit b it h has bi binding di site it ffor GDP -GDP bound non covalently Interaction of receptor with Gs-protein GDP ß GTP binds Binding site recognises GTP GTP = guanine triphosphate ß Fragmentation and release ß IInduced d d fit G-protein alters shape Complex destabilised • Process repeated for as long as ligand bound to receptor • Signal amplification - several G-proteins activated by one ligand • s Subunit carries message to next stage to make cyclic AMP © Oxford University Press, 2013 42 Signal Transduction involving Gs-Proteins Interaction of s with adenylate cyclase (inside cell) GTP GDP s-subunit b it Adenylate cyclase Binding site for s subunit GTP hydrolysed to GDP catalysed by s subunit Binding αs Induced fit ATP Active site (closed) αs P cyclic AMP Active site (open) ATP Active site (closed) Activation Protein Kinase A P Enzyme (i (inactive) ti ) cyclic AMP Enzyme ( ti ) (active) Enzyme-catalysed reaction s Subunit changes shape Weaker binding to enzyme Departure of subunit Enzyme reverts to inactive state s Subunit recombines with dimer to reform Gs protein 43 © Oxford University Press, 2013 Signal Transduction involving Gs-Proteins Interaction of cyclic AMP with protein kinase A (PKA) P t i kinase Protein ki A - 4 protein t i subunits b it - 2 regulatory subunits (R) and 2 catalytic subunits (C) cAMP C catalytic subunit released C R R R cAMP binding sites R C • Cyclic AMP binds to PKA • Induced fit destabilises complex • Catalytic units released and activated C catalytic subunit released C P Protein + ATP Protein + ADP Phosphorylation of other proteins and enzymes Signal continued by phosphorylated proteins Further signal amplification © Oxford University Press, 2013 44 Signal Transduction involving Gs-Proteins Glycogen Metabolism - triggered by adrenaline in liver cells Ad Adrenaline li signal received s s adenylate y cyclase Adrenoreceptor -Adrenoreceptor cAMP Glycogen Protein kinase A synthase (active) Catalytic C subunit of PKA Glycogen synthase-P (inactive) Phosphorylase kinase (inactive) Inhibitor (inactive) Phosphatase (inhibited) Inhibitor-P (active) Phosphorylase kinase P (active) Phosphorylase b (inactive) Phosphorylase b (active) Glycogen Glucose-1-phosphate © Oxford University Press, 2013 45 Signal Transduction involving Gs-Proteins Interaction of s with adenylate cyclase converts ATP to cAMP NH2 ATP adenosine triphosphate B P O N H O O O NH2 N O P O N O O P O O H O H O H H H B N N cyclic AMP N O enzyme control H B O P O O O P H O N O O P O O O H H H diphosphate (pyrophosphate) N O H H H B • Several 100 ATP molecules converted before s-GTP deactivated • Represents another signal amplification • Cyclic AMP becomes next messenger (secondary messenger) • Cyclic AMP enters cell cytoplasm with message (possible further amplification) © Oxford University Press, 2013 46 Signal Transduction involving Gs-Proteins Glycogen Metabolism - triggered by adrenaline in liver cells Coordinated effect: activation of glycogen metabolism ( glucose glycolysis) energy) inhibition of glycogen synthesis (no gluconeogenesis) Adrenaline has different effects on different cells e.g. activates fat metabolism in fat cells ( acetyl CoA glucose = slower) Drugs interacting with cyclic AMP signal transduction Cholera toxin causes constant activation of cyclic AMP leading to diahorrea Theophylline and caffeine -inhibit phosphodiesterases -phosphodiesterases responsible for metabolizing cyclic AMP -cyclic AMP activity is prolonged (keeps you wired) O H3C O N N O H N H3C N O CH3 N N N CH3 CH3 Theophylline Caffeine N © Oxford University Press, 2013 47 Signal Transduction involving Gs-Proteins Interaction of cyclic AMP with protein kinase A (PKA) Protein kinase A is a serine-threonine kinase Activated by cyclic AMP Catalyses phosphorylation of serine and threonine residues on protein substrates Phosphate unit provided by ATP O O H N Protein N H O B Protein H N O O P Protein O ATP N H Protein kinase A ATP ADP O O O H Protein O P ADP O O serine or threonine amino acid phosphorylated serine or threonine residue Protein kinases modify other proteins by chemically adding phosphate groups to them (phosphorylation). This usually results in a functional change of the target protein. The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes. genes Up to 30% of all human proteins may be modified by kinase activity, and kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction. © Oxford University Press, 2013 48 G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family • Rhodopsin = visual receptor • Many common receptors belong to this same family (isozymes) • Implications for drug selectivity depending on receptor similarity – consequence of evolution • Membrane bound receptors difficult to crystallise • X-Ray X Ray structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsin • Bacteriorhodopsin structure used as ‘template’ for other receptors t • Construct model receptors based on template and amino acid sequence • Leads L d tto model d l bi binding di sites it for f d drug d design i • Crystal structures for rhodopsin and 2-adrenergic receptors now solved - better templates © Oxford University Press, 2013 49 node Ethene has a pi bond. Pi bonds are second and third bonds and are generally weaker. MO diagram of a pi bond. higher, higher less stable 2pa potential energy C CC = 2pa - 2pb = antibonding MO C 2 b 2p LUMO pi-star antibond tib d C C lower, more stable HOMO CC = 2pa + 2pb = bonding MO C pi bond C LUMO = lowest unoccupied molecular orbital HOMO = highest occupied molecular orbital four C empty, but available to accept electrons Ethene MO diagram H CC = sp2a - sp2b = antibonding MO H C LUMO C H CC = p2a - p2b = antibonding MO energy of starting atomic orbitals H HOMO CC = p2a + p2b = bonding MO 63 kcal/mol 146 kcal/mol 2C = 8 e's 4H = 4 e's total bonding e's = 12 e's CC = sp2a + sp2b = bonding MO 83 kcal/mol four C full, with two electron each bond order = (12) - (0) = 6 bonds 2 © Oxford University Press, 2013 50 buta-1,3-diene - conjugated pi bonds H H H C C C H C H More nodes is less stable = no electron density between adjacent atoms. 4 C 4 = 1 - 2 H node node node node C C node node node higher, less stable C C C C 2 LUMO 1 LUMO C 3 C C C C 3 = 1 + 2 = LUMO E is larger potential energy E is smaller so is longer node 2 = 1 - 2 = HOMO 2 C llower, more stable C C 2 1 HOMO C C C C C C HOMO 1 Energy gap between HOMO and LUMO orbitals in pi bond versus diene. E = h = hc(1/) h = Plank's constant = 6.62x10-34 j-sec c = 3.0x108 m/sec C C C 1 = 1 + 2 These are constants. = frequency (#/sec = Hz) = wave length (nm = 10-9m) These are variables that indicate the energy being measured. Only one of them is needed to specify © Oxford the energy.University Press, 2013 51 The HOMO/LUMO energy gap can be measured with light (frequency = or wave length = ) systems H2C max (wave length measure of HOMO/LUMO energy gap CH2 174 nm 217 nm * 250 nm larger E (HOMO/LUMO energy gap) smaller , indicates a larger E * 280 nm E = (hc) 1 * 310 nm * 340 nm * 370 nm smaller E (HOMO/LUMO energy gap) * 400 nm * = estimated value 1 2 3 4 5 6 7 8 100 nm 200 nm 300 nm ultraviolet light 400 nm V I larger , indicates a smaller E 500 nm B G 600 nm Y 700 nm O R 800 nm 900 nm infrared light visible light IR is lower energy electromagnetic UV is higher energy electromagnetic Increasing Energy radiation that we sense as heat radiation that can damage chemical bonds -carrotene (this is the most common structure given, but there are hundreds of variations known in nature) color (?) = lycopene (found in tomatoes and other vegetables, also has hundreds of variations known in nature) color (?) = © Oxford University Press, 2013 52 higher energy LUMO E transition in UV E 100 nm 200 nm 300 nm Ultra Violet lower energy Increasing Energy 400 nm I V 500 nm 600 nm B G Y 700 nm O R 800 nm 900 nm Infrared eyes detect electromagnetic radiation (light) in this region transitions in visible -carrotene HOMO oxidation O hydration OH oxidation id ti OH rhodopsin OH recycles through vitamin A via 1. hydrolysis 2. reduction R HOD OP oxidation vitamin A H O SI N isomerization 11-trans-retinal OP Binding is weak when trans configuration. SI N protein 11-cis-retinal H N H Absorption of photon (h) causes isomerization of system to the more stable trans configuration. The release of retinal causes polarization of the cell membrane, ion channels open up and Na+ rushes in and K+ rushes out and an impulse travels to the synapse where neurotransmitters are released and complex at receptors of the next cell (neuron) which causes that cell to polarize and continue the signal to the visual part of your brain where it constructs an image that you see. binds to opsin protein R HO D OP SI N Binding is strong H when cis configuration. H N H O Aldehyde and 1o amine bind together in an imine linkage with loss of water. H 3N Rhodopsin p absorbs visible light g ((depends p on the protein charge and twist in conformation), and uses the energy to break the cis pi bond, isomerizing cis to trans, which causes a release of 11-trans-retinal from the protein and starts a cascade of reactions (cell polarization) leading to "vision". © Oxford University Press, 2013 53 How does vision work? Slightly different protein environments, changes HOMO/LUMO gap and what part of visible region gets absorbed. Your genetics determines your protein structure and the colors that you see. Different variations of protein complexes (twisting and charge distribution) absorb different wavelengths of light. E = h = h c (1 / ) What would happen if one of these was defective? E = h protein A V I = 400 nm protein C protein B B G R = red O = orange Y = yellow G = green B = blue I = indigo V = violet C B A Y O R = 700 nm complementary color wheel violet red dk blue orange lt blue yellow green yel-grn Rhodopsin complex imbedded in cell membrane has retinal bound perpendicular to 7 transmembrane protein helicies. Isomerization triggers cell action potential (polarization) and Na Na+ flows in and K K+ flows out (1 millisec). In some cells this is followed by Ca+2 ions efflux (100 millisec). y p , neurotransmitters are released from vesicles,, At a synapse, triggering the next neuron to fire and tranmit the signal to the appropriate parts of the brain cell membrane cell membrane (lipid bilayer) O AC = acetylcholine used AC in peripheral and AC AC centrall nervous systems ion channels N Na+ O vesicles with neurotransmitters fuse with cell wall and release 1000s of molecules = exocytosis Rhodopsin complex O GLU = glutamate / glutamic acid GLU important neurotranmitter GLU in the brain and spinal cord GLU O NH3 C O O HO2C NH3 DPA = dopamine used in several HO parts of the brain, cocaine and methamphetamine act on dopamine receptors, Parkinson's is caused DPA by loss of dopamine secretion HO DPA DPA cell membrane NH3 K+ Ca+2 ion channels © Oxford University Press, 2013 54 G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family Common ancestor divergent evolution Monoamines muscarinic Endothelins Bradykinin Angiotensin Interleukin-8 2 Opsins, Rhodopsins beta alpha Tachkinins Receptor types 4 5 3 Muscarinic 1 H1 H2 Histamine 1 2A 2B 2C D4 D3 D2 -Adrenergic convergent evolution D1A D1B D5 Dopaminergic 3 2 1 Receptor subtypes -Adrenergic convergent evolution 55 © Oxford University Press, 2013 G-Protein Coupled Receptors – Receptor types and subtypes Reflects differences in receptors which recognise the same ligand Receptor OH Types H HO N CH3 Adrenergic adrenoline HO O Subtypes 1, 2A, 2B, 2C Alpha Al h (()) Beta () 1, 2, 3 CH3 O acetylcholine N CH3 CH3 Cholenergic Nicotinic Muscarinic •Receptor types and subtypes are not equally distributed amongst tissues • Target selectivity leads to tissue selectivity Heart muscle Fat cells Bronchial muscle GI-tract 1 adrenergic receptors 3 adrenergic receptors 1& 2 adrenergic receptors 1 2 & 2 adrenergic receptors © Oxford University Press, 2013 56 Signal Transduction involving Gi-Proteins (i = inhibition) • Bind to different receptors from those used by Gs proteins • Mechanism of activation is identical • I subunit binds to adenylate cyclase and inhibits it • Adenylate cyclase is under dual control (brake/accelerator) • Background activity due to constant levels of s and i • Overall effect depends on dominant alpha subunit (s or i) • Dominant alpha subunit depends on receptors activated © Oxford University Press, 2013 57 Phosphorylation Reactions • Prevalent in activation and deactivation of enzymes • Phosphorylation radically alters intramolecular binding • Results R lt in i altered lt d conformations f ti NH3 NH3 NH3 might turn on, might turn off O O H O O Active site closed P O O O O O P O O O Active site open © Oxford University Press, 2013 58 Phosphorylation Reactions • Prevalent in activation and deactivation of enzymes • Phosphorylation radically alters intramolecular binding • Results R lt in i altered lt d conformations f ti CO2 OH CO2 CO2 -2 PO3 might turn on, might turn off O O P O O Active site open Active site open Active site closed © Oxford University Press, 2013 59 Signal Transduction involving Gq Proteins Interaction with phospholipase C (PLC) • Gq proteins - interact with different receptors from those recognised by GS and GI • Split by the same mechanism to give an q subunit • The q subunit activates or deactivates PLC (membrane bound enzyme) • Reaction catalysed for as long as q bound - signal amplification • Brake and accelerator effect Active site (open) Active site (closed) PLC PLC DG PLC PIP2 IP3 GTP hydrolysis DG PLC Phosphate Binding weakened PIP2 IP3 Active site (closed) q departs PLC Enzyme deactivated © Oxford University Press, 2013 60 Signal Transduction involving Gq Proteins Interaction with phospholipase C (PLC) O R R C C O H O H2 C B O C H O P O O P O OH PLC OH phospholipase p p p C O PIP2 phosphatidylinositol diphosphate O OH O OH B H P O P O O O HO H H2 C P Phosphatidylinositol diphosphate (integral part of cell membrane) R= long chain hydrocarbons P O O R R C C O O H2C C H O OH CH2 P IP3 inositol triphosphate DG diacylglycerol Inositol triphosphate (polar and moves into cell cytoplasm) Diacylglycerol (remains in membrane) = PO32© Oxford University Press, 2013 61 Signal Transduction involving Gq Proteins Action of diacylglycerol • Activates protein kinase C (PKC) • PKC moves from cytoplasm to membrane • Phosphorylates Ph h l t S Ser & Th Thr residues id off protein t i substrates bt t • Activates enzymes to catalyse intracellular reactions • Linked to inflammation, tumour propagation, smooth muscle activity etc Cell membrane DG Binding site for DG DG DG PKC Active site closed PKC Cytoplasm PKC moves to membrane Cytoplasm DG binds to DG bindingg site PKC Enzyme (inactive) Enzyme (active) Enzyme-catalysed Enzyme catalysed reaction Cytoplasm Induced fit p active site opens © Oxford University Press, 2013 62 Signal Transduction involving Gq Proteins Resynthesis of PIP2 – phosphatidylinositol diphosphate IP3 phosphatase IP2 phosphatase IP phosphatase inositol PI synthase PI PI-4-kinase PI-4-P-5-kinase PIP 2 PIP3 inhibition Li salts for manic depressive illness O O P O HO O O H O OH H H HO O O O O O R O P O OH O O O O H PIP2 O P HO H R O O P HO R O H HO H HO IP3 O O H H HO H O OH O HO H H O O P OH DG = diacylglycerol O P O H O H HO inositol R O O O 63 © Oxford University Press, 2013 Signal Transduction involving Gq Proteins Action of diacylglycerol Drugs inhibiting Protein Kinase C (PKC) - potential anti cancer agents O Me Me H O O O H Me HO OH Me O O HO O Me H O H O O H O O Me OH H Me B Bryostatin t ti (from (f sea moss)) Text, Fig 21.70 p. 566 64 © Oxford University Press, 2013 Signal Transduction involving Gq Proteins Action of inositol triphosphate •IP3 is hydrophilic and enters the cell cytoplasm • Mobilizes Ca2+ release in cells by opening Ca2+ ion channels • Ca2+ activates protein kinases Calmodulin = calcium modulated protein, • Protein kinases activate intracellular enzymes interacts with kinases and phosphatases • Cell chemistry is altered leading to a biological effect Cell membrane IP3 Cytoplasm Calmodulin Calcium stores Ca++ Calmodulin Activation Protein kinase Enzyme (inactive) Ca++ Activation P Enzyme (active) Enzyme-catalysed Enzymereaction Protein kinase Enzyme (inactive) inactive) P Enzyme (active) Enzyme-catalysed Enzymereaction © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors •Bifunctional receptor / enzyme • Activated A i db by h hormones •Protein serves dual role - receptor plus enzyme • Receptor binds messenger leading to an induced fit • Protein changes shape and opens intracellular active site • Reaction catalysed within cell • Overexpression related to several cancers messenger messenger iinduced d d fit closed active site open intracellular reaction closed 66 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors - Structure Extracellular N-terminal chain Ligand binding region NH3 H d hili Hydrophilic transmembrane region ( (-helix) Cell membrane Catalytic binding region (closed in resting state) Intracellular C-terminal chain in cytosol C O2 Tyrosine kinase 67 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors – reaction catalyzed by tyrosine kinase O O H N Protein N H Protein O O P O ATP Tyrosine kinase Mg+2 H N Protein N H Protein O O O Tyrosine amino acid H B ATP ADP O Phosphorylated tyrosine residue O P O O 68 © Oxford University Press, 2013 ADP Tyrosine Kinase Linked Receptors – Epidermal growth factor receptor (EGF- R) EGF Ligand binding & dimerisation Cell membrane Phosphorylation OH HO OH Inactive EGF-R monomers OH OP PO ATP ADP OP OP Induced fit opens tyrosine kinase active sites Binding site for EGF EGF - protein hormone - bivalent ligand Active site of tyrosine kinase Active site on one half of dimer catalyses phosphorylation of Tyr residues on other half • Dimerisation of receptor is crucial • Phosphorylated regions act as binding sites for further proteins and enzymes • Results l in i activation i i off signalling i lli proteins i andd enzymes • Message carried into cell © Oxford University Press, 2013 69 Tyrosine Kinase Linked Receptors Insulin receptor (tetrameric complex) Insulin Phosphorylation p y Cell membrane OH ATP HO OH Insulin binding site Kinase active site OH ADP PO OP OP OP Kinase active site opened by induced fit 70 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors - Growth hormone receptor Tetrameric complex constructed in presence of growth hormone GH GH binding & dimerisation Binding of kinases Activation and phosphorylation ATP GH receptors (no kinase activity) HO Kinases OH OH HO OH OH OH OH ADP PO OP OP Kinase active site opened by induced fit Growth hormone binding site Kinase active site 71 © Oxford University Press, 2013 OP Signal Transduction - Tyrosine Kinase Linked Receptors Si Signalling lli pathways th 1-TM Receptors Tyrosine kinase inherent or associated Guanylate cyclase cGMP Signalling proteins PLC IP3 DG IP3 kinase PIP3 GAP Grb2 O Others N cyclic GMP N O O P O NH2 H H O N O H Ca++ PKC H N H H © Oxford University Press, 2013 72 Signal Transduction - Tyrosine Kinase Linked Receptors Example p of a signalling g gp pathway y Growth factor 1) Binding of growth factor Dimerisation Phosphorylation 2) Conformational change HO HO HO HO OH OH HO OH OH HO PO OP PO PO PO HO OH OH OH OH OH PO OP OP OH Binding Ras and Grb2 Binding and phosphorylation of Grb2 OP PO OP PO PO PO PO Grb2 OP OP GTP/GDP exchange OP PO PO PO OP PO PO OP OP Ras GDP GTP involved in cell growth, diff differentiation ti ti andd survival i l © Oxford University Press, 2013 73 Signal Transduction - Tyrosine Kinase Linked Receptors Example p of a signalling g gp pathway y Growth factor 1) Binding of growth factor Dimerisation Phosphorylation 2) Conformational change HO HO HO HO OH OH HO OH OH HO PO OP PO PO PO HO OH OH OH OH OH PO OP OP OH Binding Ras and Grb2 Binding and phosphorylation of Grb2 OP PO OP PO PO PO PO Grb2 OP OP GTP/GDP exchange OP PO PO PO OP PO PO OP OP Ras GDP GTP involved in cell growth, diff differentiation ti ti andd survival i l © Oxford University Press, 2013 74 Signal Transduction - Tyrosine Kinase Linked Receptors Example of a signalling pathway Ras OP PO OP PO PO PO PO OP Gene transcription OP Raf (inactive) Raf (active) Mek (inactive) Mek (active) Map kinase (inactive) Map kinase (active) Transcription factor (inactive) Transcription factor (active) © Oxford University Press, 2013 75 Intracellular Receptors - Structure • Chemical messengers must cross cell membrane • Chemical messengers must be hydrophobic • Example - steroids and steroid receptors CO2 Steroid binding region Zinc DNA binding region (‘zinc fingers’) H3N Zinc fi Zi fingers contain t i C Cys residues id (SH) Allow S-Zn interactions with proteins that bind to DNA and RNA © Oxford University Press, 2013 76 Intracellular Receptors – Mechanism Co-activator protein Receptor DNA Messenger Receptor-ligand complex Cell membrane 1. 2. 3. 4. 5. 6. 7 7. Dimerisation Messenger crosses membrane Binds to receptor Receptor p dimerisation Binds co-activator protein Complex binds to DNA Transcription switched on or off P t i synthesis Protein th i activated ti t d or inhibited i hibit d © Oxford University Press, 2013 77 Intracellular Receptors – Estrogen receptor Binding site AF-2 g regions H12 Coactivator Coactivator Estradiol DNA Estrogen receptor Dimerisation & exposure of AF-2 regions CH3 Nuclear transcription factor Transcription OH CH3 H H CH3 H H H OH How? H H H HO O Testosterone Estradiol / Estrogen © Oxford University Press, 2013 78 The oxidation of testosterone to make estradiol shows yet another way of losing a methyl group through a conjugated C=C bond. Methyl is removed and an aromatic ring is formed. formed OH CH3 CH3 CH3 H H H H H testosterone a male sex hormone O H2C O H C N +3 Fe +4 O O B H H H O O R B H H H O H N O NADH H R R N R NAD+ oxidation O O B O H H O C O N B FADH2 H H H O B H O O H B N R H dehydrogenation B H d decarboxylation b l ti R tautomers O R C FAD H B R H N N H R H C C NAD+ oxidation O R Fe B H R CH2 O R sp3 C-H oxidation O H CH2 Fe +4 H -estradiol a female sex hormone HO R B H HO H O OH H O © Oxford University Press, 2013 79 Overview of signal transduction pathways 80 © Oxford University Press, 2013 Signal transduction occurs when an extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell. That receptor triggers a biochemical chain of events inside the cell. Depending on the cell, the response alters the cell's metabolism, shape, gene expression ability to divide and more. expression, more The signal can be amplified at any step. step G protein-coupled receptors integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. (includes adrenergic receptors and chemokine receptors.) Tyrosine kinase receptors are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; (includes growth factor receptors such as the insulin receptor) Integrins play a role in cell attachment to other cells and the extracellular matrix and in the transduction of signals from extracellular matrix components such as fibronectin and collagen. Toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation. A ligand-gated ion channel, upon binding with a ligand, changes conformation to open a channel in the cell membrane through which ions relaying signals can pass. An example of this mechanism is found in the receiving cell of a neural synapse. Intracellular receptors, receptors such as nuclear receptors and cytoplasmic receptors, receptors are soluble proteins localized within their respective areas. The typical ligands for nuclear receptors are non-polar hormones like the steroid hormones testosterone and progesterone and derivatives of vitamins A and D. To initiate signal transduction, the ligand must pass through the plasma membrane by passive diffusion. On binding with the receptor, the ligands pass through the nuclear membrane into the nucleus, altering gene expression. i N l i receptors Nucleic t have h DNA bi di domains DNA-binding d i containing t i i zinc i fingers fi and d a ligand-binding li d bi di domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. Second messengers: Ca+2, lipophilics (diacylglycerol), nitric oxide and other redox signaling molecules 81 © Oxford University Press, 2013