Chaps 4 & 5: Receptors as drug targets, structure, function &

Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates 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 is crucial for treating diseases effectively. signals neighbor receptors () self talk talk cell 1 signals cell 2 cell 1 cell 2 cell 3 etc. synapse synapse synapse signals signals a () (hormones) receptor b cell 2 cell 3 The same signal can deliver different cell 1 messages to different tissues (histamine). cell 5 cell 4 receptor c signals signals (hormones) (hormones) receptor d Important functions of receptors: 1. Globular (receptors) acting as a cell’s ‘letter boxes’ 2. Located mostly in the cell membrane 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 1 © Oxford University Press, 2013 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 = signal S R R = receptor Autocrine signals are produced by the target cell, are secreted, and affect the target cell itself via receptors. Sometimes autocrine cellscantarget cellsclose by if they arethe same type of cell as the 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 or lipid components integral to the membrane and are capable of affecting either the emitting cell or cells immediately adjacent. R S cell a R cell b Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.

S R cell a cell b S R cell c S etc. synapse synapse 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 S R2 cell c S S S S cell a S S S S S S S R3 cell d R1 cell b 2 © Oxford University Press, 2013 Central Nervous System (CNS) structure Brain and spinal cord Integrative and control centers, function process info out and info in

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 Motor nerve fibers nerve fibers Conducts impulses from Conducts impulses from the CNS receptors to the CNS to effectors (muscles and glands)

Sympathatic division Autonomic nervous system (ANS) Somatic nervous system mobilizes body systems during activity (fight or flight) Visceral motor (involuntary) Somatic motor (voluntary) Conducts impulses from the CNS to Conducts impulses from the cardiac muscles, smooth muscles, CNS to skeletal muscles Parasympathetic division and glands Conserves energy Promotes 'housekeeping' functions during rest 3 © Oxford University Press, 2013 Central nervous system The central nervous system consists of the two major structures: the brain and spinal cord. The brain is encased in the skllkull, and prot ect tdbthed by the crani um.

The spinal cord is continuous with the brain and lies on the backside to the bibrain, and di is prot ttdbthected by the vert tbebra. The spinal cord reaches from the base of the skull, continues through or starting below the foramen magnum, and termi nat es roughl y l evel with th e fi rst or second lumbar vertebra, occupying the upper sections of the vertebral canal.

The CNS in tegra tes in forma tion it receives from, and coordinates and influences the activity of, all parts of the body and it contains the majority of the nervous system. U suall y, th e reti na and the optic nerve (2nd cranial nerve), as well as the olfactory nerves (1st) and olfactory epithelium are considered as parts of the CNS, s ynapsing directly on brain tissue without intermediate ganglia.. 4 © Oxford University Press, 2013 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 . 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,,(),,g lacrimation (tears), urination, digestion and defecation. Its action is described as bein 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

6 © Oxford University Press, 2013 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 Somatic nervous system (toward) and efferent nerves (out 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 .

The somatic nervous system consists of three parts:

Spinal nerves: They are peripheral nerves that carry sensory ifinformati on i nto and motor command s out of fh the spinal cord.

Cranial nerves: They are the nerve fibers that carry information into and out of the brain stem. They include smell, vision, eye, eye muscles, mouth, taste, ear, neck, shoulders, and tongue.

Interneurons (association nerves): These nerves integrate sensory input and motor output , numbering thousands

7 © Oxford University Press, 2013 Nerve cells make contact to muscle cells, organs and other nerve cells

Can be 1000s of axon inputs from other nerve cells.

… or synaptic button when contact to neuron

8 © Oxford University Press, 2013 axon

Synaptic button or neuromuscular junction

Cell body

9 © Oxford University Press, 2013 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), here ditary sensor imo tor neuropath y and peroneal muscul ar at roph y, i s a h et erogeneous 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 neurologi cal di sord ers, with 37 i n 100 ,000 aff ect ed ( 1 i n 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 bhbehav iora l c hanges. ThdiThe disease process is assoc itdiated with ithl plaques and dt tangl es i n th thbe brai n. Th e 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 extend s t o th e d omai ns of l anguage, skill ed movement s and recogniti on, and f uncti ons such as decision-making and planning become impaired.

Parkinson's disease (PD), is a degenerative disorder of the central nervous system that often impai rs th e suff erer' s mot or skill s an d speec h. PkiParkinson' 'dis disease b blelongs t o a group of 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 btheby the insufficient formation and action of dopamine , which is prod uced 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. 11 © Oxford University Press, 2013 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 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 autonomic nervous system. Symptoms usually develop gradually over years. 12 © 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, starting the next impulse. neurotransmitters bind to receptors on the next cell at the synapse depolarization Some of these are neurotransmitters milli seconds at synapse activated by the change in membrane potential. Axon proteins neurotransmitters leave cell by exocytosis 0 +2 action Ca K Na Cl potential action potential K

Na Na Cell Body Ca+2

Ion gate opens when K Na Ca+2 neurotransmitter Cl +2 H Ca N complexes dendrites O at receptor. O H B H B O choline retaken up by O presynaptic neuron N O O H O OH N H B O diffuses across the synaptic O O H O acetyl choline cleft and binds to a Na+ H H B is an important B neurotransmitter, ion-gate channel on the B BH OH of a serine residue of the receptor receptor receptor receptor hydrolyzed back to the serine "OH" 13 © Oxford University Press, 2013 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, , and proteins). The glands that secrete hormones comprise the endocrine signaling system. The term 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 H pre-vitamin D3

HO

H

H

vitamin D3

17 HO © Oxford University Press, 2013 1. Insulin binds to tyrosine Hormone binding to receptor - insulin kinase-linked receptor. 2. Phosphorylation of receptor, starts seqqguence leading to (Insulin protein requires post translational modification) synthesis of glycogen, storage of glucose (and fat too) 3. Allows influx of glucose 4. Insulin-like molecules are even found in the simplest unicellular eukaryotes, over 1 billion years old 5. Our insulin is very similar to pig and cow insulin, was used before our genes were sppgyliced into genetically modified bacteria

(glucose transport)

http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cfm 18 © Oxford University Press, 2013 Icosanoids are signaling molecules made by oxidation of either 20-carbon omega-3 (-3) or omega- 6 (-6) fatty acids. In general, the -6 eicosanoids are pro-inflammatory; -3s are much less so. There are multiple subfamilies of eicosanoids, including the prostaglandins, 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 , 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 's diet will affect the body 's icosanoid-controlled functions, with effects on cardiovascular disease, triglycerides, blood pressure, and arthritis.

prostaglandins thromboxanes O leukotrienes O O O O OH OH OH OH

HO OH O OH lipoxin B4 - eoxin, pro- alprostadil - inhibits thromboxane A2 - vasoconstrictor, imfalmmatory (white blood cells), blood clotting , vasodilator hypertensive, blood clotting, mast cells, contributes to allergies, aspirin inhibitsitsformation various cancers, Hodgkin's lymphoma

19 © Oxford University Press, 2013 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-inflammatory drug.Steroidshavetwo 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.

glucocorticoids (adrenal cortex) C D mineralocorticoids (adrenal cortex) estrogens (gonads) androgens (gonads) A B progestins (ovaries and placenta) vitamin D (diet and sun) ...and more Letters are used to identify the 4 rings.

20 © Oxford University Press, 2013 OH CH3 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 levels in men are lower compared towomen,estrogens have essential functions inmenaswell. It is 2 steps away from progesterone. OH CH3 H

CH3 H

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 development of male reproductive tissues such as thetestisand prostate as well as promoting secondary 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 H H C O Common steroid transformations V22, p. 703, p 1263 3 H3C CH CH while similar in appearance, 3 C 3 CH3 CH everyone of these steroids has a H 3 H H specific signal to transmit, such as CH 3 CH H ? steps turning on a genetic switch. They 3 CH3 H are extremely powerful chemicals, about 19 steps CH3 some operating at the nanomolar HH H H level. HO lanosterol - the compound HO cholesterol - essential component pregnenolone - H from which all steroids are of cell walls, precursor of steroid HO neuroactive steroid H3C CH3 derived. hormones, bile acids and vit D sp3 oxidation NAD+ and isomerization O O O CH CH C 3 CH 3 CH 3 CH C 3 CH C 3 3 H OH OH NAD+ and CH H CH H 3 CH H isomerization 3 sp3 oxidation squalene oxide 3 H H H H O H H progesterone - involved O O in menstrual cycle and HO pregnacy 17-hydroxypregnenolone - higher levels are 17-hydroxyprogesterone - sometimes found at end of puberty and in pregnacy. given to prevent premature birth 3 OH 3 OH sp oxidation O ? steps sp oxidation O CH2 CH2 C O C CH3 CH3 CH3 H squalene OH

CH3 H CH3 H CH3 H Statins drugs work here. H H H H H H

O O 11-deoxycorticosterone O HO O intermediate to aldosterone O O dehydroisoandrosterone - most abundant P P O 11-deoxycortisol (cortodoxone) O circulating steroid, many uses glucocorticoid (similar to cortosol) 3 O sp oxidation OH O OH O dimethylallyl isopentenyl NADH O OH 3 CH2 phosphate CH3 sp oxidation C phosphate CH2 CH3 CH C H 3 HO H HO OH CH3 H CH3 H CH3 H many steps H H reduced C=C is H H H H O O (maybe with FADH2) cortisol - glucocorticoid, increases O corticosterone - stress acetyl CoA testosterone - produced (more potent by 3x) O blood sugar, decreases immune CoA system (hydrocortisone) 3 hormone, immune system S in all vertebrates, 20x more sp oxidation in males, male sex characteristics. and OH three well known members of the estrogen family O 5 categories of steroid hormones OH O NAD+ oxidation O H CH OH CH2 1. progestins - overies and placenta- mediate see 3 CH3 C reactions CH3 C menstrual cycle and maintains pregnacy HO 2. glococorticoids - adrenal cortex - affect below OH H H metabolism in diverse ways, decrease H OH CH H inflamation,incease resistance to stress 3 3. mineralocorticoids - adrenal cortex - maintain salt and water balance H H H H H H H H 4. androgens - gonads - maturation and function NAD+ of secondary sex organs, particularly in males, HO 3 HO -estradiol - prominant estrogen HO sp oxidation oxidation O aldosterone - Na+,K+ male sexual differentiation estrone - known female balance, blood pressure reg. 5. estrogens - gonads - maturation and function of in reproductive years, more potent estriol - higher levels in pregnancy, carcinogen, causes breast tenderness, secondary sex organs, particularly in females than estriol (10x) and estrone (80x) marker of fetal health in men causes anorexia 22 © Oxford University Press, 2013 Amino acid derivatives (or from amino acids) – thyroxine, histamine, catacholamines (epinephrine, norepinephrine) oxytocin, , glutamate, glycine, gama aminobutyric acid, acetylcholine (neurotransmitters and hormones)

HO O O HO H2 HO N HO NH3 O O

NH epinephrine glutamate 3 HO adrenaline HO norepinephrine

I O O O HO I

H3N O H3N O O -aminobutyric acid NH3 glycine I O

thyroxine (T4) I HO O N O NH 3 N N HO NH3 HN nitrosyl O radical histamine dopamine (nitric oxide) acetylcholine Cys S Tyr oxytocin - 8 amino acid S

Gly Leu Pro Cys Asn Ile 23 © Oxford University Press, 2013 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 receppg,tor on a target cell, such as a muscle cell or another neuron. Usuall 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 receppgtors on target cells throu ghout the bod y

Chemical messengers ‘switch on’ receptors without undergoing a reaction (different from enzymes)

Nerve 1

Synaptic button =, connections to neurons, acetylhlilcholine receptors Neuromuscular end plates = connections to muscles, adrenoline Blood supply , e. g. insulin – absorb glucose Nerve 2 Glucagon – release glucose Hormone response Cell

Neurotransmitters secondary messenger 24 © Oxford University Press, 2013 Structure and function of receptors Mechanism

Messenger Induced fit Messenger

Messenger

Cell Membrane Receptor Receptor Receptor

Cell Cell Cell Mmessageessage

Receptors contain a binding site (hydrophobic hollow or cleft on the receptor protein) that is recognised by the chemical messenger ion - ion H bonds Binding of the messenger involves intermolecular bonds with amino acids 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) 25 © Oxford University Press, 2013 Binding interactions • Ionic • H-bonding • van der Waals

Induced fit - Binding site alters shape to maximise intermolecular bonding

Phe Phe

H O H O Ser Ser CO2 CO2 Induced Asp Fit Asp

Intermolecular bonds not optimum Intermolecular bond lengths optimised, length for maximum binding strength change of shape causes a change in 26 function (on or off)© Oxford University Press, 2013 Overall Process of Receptor/Messenger Interaction

M M M = messenger M R = receptor

RE R RE

Signal transduction

Bindi ng i nteracti ons must b e strong enough to h old th e messenger suffi ci entl y l ong 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) 27 © Oxford University Press, 2013 Receptor Superfamilies RESPONSE TIME • RECEPTORS msecs

•G-PROTEIN COUPLED RECEPTORS MEMBRANE seconds BOUND •KINASE LINKED RECEPTORS 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

29 © Oxford University Press, 2013 Ion Channel Receptors - General principles hydrophilic tunnel

Cell membrane

hydrophobic exterior

MESSENGER Extra cellular Extra cellular

ION ION RECEPTOR CHANNEL CHANNEL BINDING (closed) ()(open) SITE MESSENGER

Induced fit and opening Lock Gate of ion channel Cell Ion Ion Cell Cell Ion Ion Cell membrane channel channel membrane membrane channel channel membrane

Cell Cell

30 © Oxford University Press, 2013 Ion Channel Receptors - Gating

Binding site Receptor Messenger

Induced Cell Cell membrane fit 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 receptor subunits)

Neurotransmitter binding region  

H2N Extracellular loop   one of Cell  these membrane CO2H

Cell TM1 TM2 TM3 TM4 membrane

Intracellular Variable loop loop

4 Transmembrane (TM) regions (hydrophobic on the outside of channel)

32 © Oxford University Press, 2013 Ion Channel Receptors - Detailed Structure of Ion Channel

TM4 Protein

TM1 TM3 subunits

TM3 TM2 TM1

TM4 TM2 TM2 TM4

TM1 TM3

TM3 TM2 TM2 TM1 Transmembrane

TM4TM1 TM3 TM4 regions

Note: TM2 of each protein subunit ‘lines’ the central pore (TM2 is more polar, perhaps bonded to very polar carbohydrates, TM1, TM33d and TM4 ar eoeydopobcdceowdsceebe.)e more hydrophobic and face towards cell membrane.)

33 © Oxford University Press, 2013 Ion Channel Receptors - Gating Ion flow

TM2 TM2 Cell membrane

TM2 TM2

TM2 TM2 TM2 TM2 Transverse view Transverse view TM2 TM2 TM2

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

34 © Oxford University Press, 2013 Ion Channel Receptors – Nicotinic receptor

Bindi ng sites

Ion channel    

 Cell     membrane 

Two binding sites x subunits mainly on -subunits

CH3 O H3C H3C CH3 N N CH3 N H3C O H3C O CH3 H CH3

acetylcholine nicotine neurotransmitter protonated at body 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. 35 © Oxford University Press, 2013 Ion Channel Receptors – Glyypcine receptor

Binding sites

Ion channel     Cell    membrane   

Three ligand binding sites xx subunits on --subunitssubunits

O 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 H3N O nervous system and has important roles in a variety of physiological processes, glycine especially in mediating inhibitory in the spinal cord and brainstem.

36 © Oxford University Press, 2013 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,,p is destabilised then split  messenger idinduced fit

closed open

G-protein split into α and β/ subunits

37 © Oxford University Press, 2013 G-Protein Coupppled Receptors – General ppprinciples • 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 active site (closed) ()(open)

ItIntracell lllular reaction 38 © Oxford University Press, 2013 G-PtiCProtein Coupl ldRed Recept ors - Struc ture Single protein with 7 transmembrane regions (7-TM receptor)

Extracellular loops NH3 extracellular N -Terminal chain

Transmembrane Membrane VII VI V IV III II I helix

G-Protein binding region cytoplasm O C 2 Variable Intracellular loops C -Terminal chain intracellular loop

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 BDC

A) Monoamines: pocket in TM helices B) Peptide hormones: top of TM helices + extracellular loops + N-terminal chain C) Hormones: ex trace llul ar l oops + N-tilhiterminal chain 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 - thyy,roxine, testosterone, ,g,y,pp estrogen, oxytocin, epinephrine • Glutamate, DOPA, glycine, acetylcholine, Ca++

40 © Oxford University Press, 2013 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 splitsoff G protein-coupled and binds to another receptors (7 TM receptors) protein to start signal imbedded in membranes cascade inside the cell  continuing  various reactions protein   actions inside cell protein inside cell 41 © Oxford University Press, 2013 DRUG TARGETS SIGNAL TRANSDUCTION

Signal Transduction involving Gs-Proteins provide one step in signal transduction pathway.

GS--ProteinProtein - membrane bound protein of 3 subunits () - S subun it has bindi ng sit e for GDP -GDP bound non covalently    Interaction of receptor with Gs-protein GDP

ß ß   GTP binds Fragmentation   and release  ß  IdInduce dfitd fit Binding site recognises GTP G-protein alters shape GTP = guanine triphosphate 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 42 © Oxford University Press, 2013 Signal Transduction involving Gs-Proteins GTP s--susubitbunit Interaction of s with adenylate cyclase (inside cell) GDP Adenylate cyclase

Binding site for s subunit GTP hydrolysed to GDP catalysed

Binding by s subunit Induced fit αs P αs ATP cyclic AMP ATP cyclic AMP Active site Activation Active site Active site (open) (closed) (closed) Protein s Subunit changes shape Kinase A Weaker binding to enzyme P Departure of subunit Enzyme Enzyme Enzyme reverts to inactive (inacti ve) (ti)(active) state

Enzyme-catalysed s Subunit recombines with  dimer to reform Gs protein 43 reaction © Oxford University Press, 2013 Signal Transduction involving Gs-Proteins Interaction of cyclic AMP with protein kinase A (PKA) PtikiProtein kinase A - 4tibit4 protein subunits - 2 regulatory subunits (R) and 2 catalytic subunits (C) cAMP C catalytic subunit released C R R

R R cAMP C binding sites • Cyclic AMP binds to PKA C • Induced fit destabilises complex catalytic subunit released • Catalytic units released and activated

C Phosphorylation of other proteins and enzymes P Signal continued by phosphorylated proteins Further signal amplification Protein Protein + ATP + ADP

44 © Oxford University Press, 2013 Signal Transduction involving Gs-Proteins Glycogen Metabolism - triggered by adrenaline in liver cells Adrenaline signal received s s

-Adrenoreceptor adenylate cyclase cAMP

Glycogen Protein kinase A synthase Inhibitor (active) (inactive) Catalytic C subunit of PKA Glycogen Inhibitor-P Phosphatase synthase-P (active) (inhibited) (inactive)

Phosphorylase Phosphorylase kinase (inactive) kinase P (active)

Phosphorylase b Phosphorylase b (inactive) (active)

Glycogen Glucose-1-phosphate 45 © Oxford University Press, 2013 Signal Transduction involving Gs-Proteins

Interaction of s with adenylate cyclase converts ATP to cAMP

NH2 ATP NH2 adenosine triphosphate N N B BH N N cyclic AMP O O O N N O O H O N N O P O P O P O O O P O P O enzyme O P O O O O H H control O O O H H H H O H H H diphosphate O H H B (pyrophosphate) 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)

46 © Oxford University Press, 2013 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 O H CH3 H C H3C N 3 N N N

N N O N O N

CH3 CH3 Theophylline Caffeine 47 © Oxford University Press, 2013 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 H N N Protein O Protein Protein N Protein N H H O P O ATP Protein kinase A O ADP O O O B H O P O ATP ADP O serine or threonine phosphorylated serine amino acid 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 gene sandtheys and they constitute about 2% of all human 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. 48 © Oxford University Press, 2013 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 structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsin • Bacteriorhodopsin structure used as ‘template’ for other receptors • Construct model receptors based on template and amino acid sequence • LdtLeads to mo dlbidiitfdel binding sites for drug didesign

• Crystal structures for rhodopsin and 2-adrenergic receptors now solved - better templates

49 © Oxford University Press, 2013 Ethene has a pi bond. Pi bonds are second node and third bonds and are generally weaker. MO diagram of a pi bond.   = 2p - 2p = antibonding MO pi-star CC a b C C higher, 2pa LUMO 2pb antibond less stable

potential energy C C

lower, more stable pi bond  = 2p + 2p = bonding MO CC HOMO CC a b LUMO = lowest unoccupied molecular orbital HOMO = highest occupied molecular orbital

Ethene MO diagram four C empty, but available to accept electrons

2 2 H H CC = sp a - sp b = antibonding MO C 2 2 LUMO CC = p a - p b = antibonding MO C energy of starting H H atomic orbitals

2 2 HOMO CC = p a + p b = bonding MO  63 kcal/mol 2C = 8 e's  146 kcal/mol 4H = 4 e's  = sp2 + sp2 = bonding MO  83 kcal/mol total bonding e's = 12 e's CC a b

four C full, with two electron each

(12) - (0) = 6 bonds 50 bond order = 2 © Oxford University Press, 2013 More nodes is less stable = no buta-1,3-diene - conjugated pi bonds electron density between adjacent atoms. node node node H H

H C C C C H 4 CC CC 4 = 1 - 2 H H node node node node

higher, CC CC less stable  1 2 CC LUMO LUMO 3 CC

3 = 1 + 2 = LUMO

E is E is smaller node so  is longer potential larger energy 2 = 1 - 2 =HOMO 2 CCC CC

C C C C lower, 2 more stable 1 HOMO HOMO

1 CCC CC Energy gap between HOMO and  =  +  LUMO orbitals in pi bond versus diene. 1 1 2

E = h = hc(1/) These are variables that indicate  = frequency (#/sec = Hz) the energy being measured. Only -34 These are h = Plank's constant = 6.62x10 j-sec  = wave length (nm = 10-9m) one of them is needed to specify 8 constants. 51 c = 3.0x10 m/sec the© Oxfordenergy. University Press, 2013 The HOMO/LUMO energy gap can be measured with light (frequency =  or wave length = )  systems max (wave length measure of HOMO/LUMO energy gap

H2CCH2 174 nm larger E (HOMO/LUMO energy gap) 217 nm smaller , indicates a larger E 250 nm*

280 nm* E = (hc) 1  310 nm*

* 340 nm larger , indicates a smaller E 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 400 nm 500 nm 600 nm 700 nm 800 nm 900 nm V I B G Y O R ultraviolet light infrared light

visible light UV is higher energy electromagnetic IR is lower energy electromagnetic radiation that can damage chemical bonds Increasing Energy radiation that we sense as heat -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 (?) =

52 © Oxford University Press, 2013 higher energy Increasing Energy lower energy

 100 nm 200 nm 300 nm 400 nm 500 nm 600 nm 700 nm 800 nm 900 nm LUMO Ultra Violet V I B G Y OR Infrared

E eyes detect electromagnetic E transition transitions in visible radiation (light) in this region in UV -carrotene  HOMO

oxidation

O

hydration OH

oxidation OH OH rhodopsin

recycles through vitamin A via oxidation vitamin A 1. hydrolysis 2. reduction H

R H ODO O P S I N isomerization 11-trans-retinal

Binding is weak when O protein P S trans configuration. I N 11-cis-retinal H binds to opsin protein N o R HO D Aldehyde and 1 amine O P H O H S bind together in an imine I N Absorption of photon (h) causes linkage with loss of water. isomerization of  system to the H3N more stable trans configuration. Rhodopsin absorbs visible light (depends The release of retinal causes polarization of the cell on the protein charge and twist in membrane, ion channels open up and Na+ rushes in and conformation), and uses the energy to break K+ rushes out and an impulse travels to the synapse the cis pi bond, isomerizing cis to trans, where neurotransmitters are released and complex at which causes a release of 11-trans-retinal receptors of the next cell (neuron) which causes that from the protein and starts a cascade of cell to polarize and continue the signal to the visual part Binding is strong H N reactions (cell polarization) leading to of your brain where it constructs an image that you see. when cis configuration. "vision". 53 H © Oxford University Press, 2013 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 complementary of these was defective? color wheel

R = red E = h violet A B C O = orange red Y = yellow protein protein protein G = green dk blue orange A B C B = blue yellow I = indigo lt blue V = violet green yel-grn V I B G Y O R  = 400 nm  = 700 nm Rhodopsin complex imbedded in cell membrane has retinal bound perpendicular to 7 transmembrane protein helicies. Isomerization At a synapse, neurotransmitters are released from vesicles, triggers cell action potential (polarization) and Na+ flows in and K+ triggering the next neuron to fire and tranmit the signal to flows out (1 millisec). In some cells this is followed by Ca+2 ions the appropriate parts of the brain efflux (100 millisec). cell membrane cell membrane (lipid bilayer) O AC AC = acetylcholine used Na+ AC in peripheral and ion channels AC central nervous systems N O

vesicles with neurotransmitters Rhodopsin fuse with cell wall and release complex 1000s of molecules = exocytosis O O NH3 GLU GLU = glutamate / glutamic acid C HO2C GLU important neurotranmitter O O GLU in the brain and spinal cord NH3

DPA = dopamine used in several HO NH parts of the brain, cocaine and 3 methamphetamine act on dopamine receptors, Parkinson's is caused DPA by loss of dopamine secretion HO K+ DPA Ca+2 DPA ion channels cell membrane 54 © Oxford University Press, 2013 G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family

Common ancestor divergent evolution

Monoamines muscarinic Opsins, beta alpha Rhodopsins

Bradykinin Endothelins Tachkinins Interleukin-8 Receptor types

Receptor 2 4 5 31H1H21 2A 2B 2C D4 D3 D2 D1A D1B D5 3 2 1 subtypes

Muscarinic Histamine -Adrenergic Dopaminergic -Adrenergic

convergent evolution 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 Types Subtypes OH H HO N Alph a () CH3 Adrenergic 1, 2A, 2B, 2C adrenoline Beta () HO 1, 2, 3

O CH3 CH3 N Nicotinic O CH3 Cholenergic acetylcholine Muscarinic 

•Receptor types and subtypes are not equally distributed amongst tissues • Target selectivity leads to tissue selectivity

Heart muscle 1 adrenergic receptors Fat cells 3 adrenergic receptors Bronchial muscle 1& 2 adrenergic receptors GI-tract 1 2 & 2 adrenergic receptors 56 © Oxford University Press, 2013 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

57 © Oxford University Press, 2013 Phosphorylation Reactions • Prevalent in activation and deactivation of enzymes • Phosphorylation radically alters intramolecular binding • Resu lts i n alt ered conf ormati ons

NH3 NH3 NH3

O might turn on, O P O might turn off

O O O H O P O O O O O

Active site Active site open closed

58 © Oxford University Press, 2013 Phosphorylation Reactions • Prevalent in activation and deactivation of enzymes • Phosphorylation radically alters intramolecular binding • Resu lts i n alt ered conf ormati ons

CO2 CO2 CO2

-2 OH PO3 might turn on,

O might turn off O P O O

Active site Active site Active site open open closed

59 © Oxford University Press, 2013 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 Active site (open) (closed) DG    PIP PLC PLC PLC 2

IP3

Active site GTP hydrolysis  departs (closed) DG q  PIP  PLC 2 PLC

IP Phosphate 3 Enzyme Binding deactivated weakened 60 © Oxford University Press, 2013 Signal Transduction involving Gq Proteins Interaction with phospholipase C (PLC) R R

O C C O H B O O H HO O H2 H C C C P 2 H O O O O R R H B O P P OH O O C C O O O P OH O O OH O OH PLC OH H C C CH2 2 H phospholipase C O P IP3 DG PIP2 O inositol triphosphate diacylglycerol phosphatidylinositol diphosphate P Phosphatidylinositol diphosphate Inositol triphosphate Diacylglycerol (integral part of cell membrane) (polar and moves (remains in into cell cytoplasm) membrane)

2- R= long chain hydrocarbons P = PO3

© Oxford University Press, 2013 61 Signal Transduction involving Gq Proteins Action of diacylglycerol • Activates protein kinase C (PKC) • PKC moves from cytoplasm to membrane • Phosph ory lat es Ser & Thr res idues o f pro te in su bs tra tes • Activates enzymes to catalyse intracellular reactions • Linked to inflammation, tumour propagation, smooth muscle activity etc

Cell membrane DG Binding DG site for DG DG PKC PKC

Enzyme Enzyme (inactive) (active) PKC Active site closed Enzyme-catalysed reaction Cytoplasm Cytoplasm Cytoplasm PKC moves DG binds to Induced fit to membrane DG binding site opens active site

62 © Oxford University Press, 2013 Signal Transduction involving Gq Proteins

Resynthesis of PIP 2 – phosphatidylinositol diphosphate phosphatase phosphatase phosphatase IP3 IP2 IP PI synthase PI-4-kinase PI-4-P-5-kinase inositol PI PIP3 PIP2

inhibition

Li salts for O manic depressive illness R O

O R O IP H HO PIP O 3 HO H O 2 O P O HO OH O O O H P O P O O O H HO O O H H P O HO O OH HO H HO H HO O OH H H O inositol HO R O O H H H O R P O H O O O O P O OH O DG = diacylglycerol 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 Me O H O

HO O O O H Me HO Me OH H O O H

O O

H O Me OH O H Me Bryos ta tin (f rom 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 2+ • Ca 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

IP 3 Cytoplasm

Calmodulin Calcium Ca++ Calmodulin Ca++ stores Activation Activation Protein Protein kinase kinase P P

Enzyme Enzyme Enzyme Enzyme (inactive) (active) (inactive) (active)

EnzymeEnzyme--catalysedcatalysed EnzymeEnzyme--catalysedcatalysed reaction © Oxfordreaction University Press, 2013 Tyrosine Kinase Linked Receptors •Bifunctional receptor / enzyme • AiActivate dbhd by 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

idinduced fit

active site closed closed open

intracellular reaction 66 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors - Structure

Ligand binding region Extracellular N H 3 N-terminal chain

Hyd rop hilic transmembrane Cell membrane region (--helix)helix)

Catalytic binding region (closed in resting state) Intracellular C-terminal CO2 chain in cytosol

Tyrosine kinase 67 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors – reaction catalyzed by tyrosine kinase

O O H H N N Protein O Tyrosine Protein Protein N Protein N H kinase H O P ATP O ADP O Mg+2 O

ATP ADP O O

Tyrosine O P O amino acid H Phosphorylated tyrosine residue B O

68 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors – Epidermal receptor (EGF- R) EGF

Ligand binding Cell & dimerisation Phosphorylation membrane

HO OH PO OP OH OH ATP ADP OP OP Inactive EGF-R Induced fit monomers 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 i n acti vati on of si gnalli ng protei ns and enzymes • Message carried into cell 69 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors Insulin receptor (tetrameric complex)

Insulin

Phosppyhorylation Cell membrane ATP HO OH ADP PO OP OH OH OP OP Kinase active site Insulin binding site opened by induced fit Kinase active site

70 © Oxford University Press, 2013 Tyrosine Kinase Linked Receptors - Growth hormone receptor Tetrameric complex constructed in presence of growth hormone

GH

GH binding & Binding Activation and dimerisation of kinases phosphorylation

ATP ADP GH receptors (no kinase activity) HO OH PO OP Kinases OH OH OP OP

HO Kinase active site OH OH OH opened by induced fit Growth hormone binding site Kinase active site

71 © Oxford University Press, 2013 Signal Transduction - Tyrosine Kinase Linked Receptors

Signalli ng path ways 1-TM Receptors

Tyrosine kinase inherent or associated Guanylate cyclase

Signalling proteins cGMP

O PLC IP3 GAP Grb2 Others N H kinase N cyclic GMP

N O N NH2 IP DG PIP 3 3 O P O O H H ++ H Ca PKC H O H 72 © Oxford University Press, 2013 Signal Transduction - Tyrosine Kinase Linked Receptors Examppggpyle of a signalling pathway

Growth factor

1) Binding of growth factor Dimerisation Phosphorylation 2) Conformational change HO HO HO HO PO PO OH OH OH OH OP OP HO OH PO PO HO OH HO OH OH OH PO OP

OH Binding Grb2 Ras and Ras GDP Binding and OP GTP/GDP OP GTP Grb2 exchange phosphorylation PO PO PO PO OP of Grb2 OP OP OP involved in cell growth, PO PO PO OP PO PO PO OP differenti ati on and survi val

73 © Oxford University Press, 2013 Signal Transduction - Tyrosine Kinase Linked Receptors Examppggpyle of a signalling pathway

Growth factor

1) Binding of growth factor Dimerisation Phosphorylation 2) Conformational change HO HO HO HO PO PO OH OH OH OH OP OP HO OH PO PO HO OH HO OH OH OH PO OP

OH Binding Grb2 Ras and Ras GDP Binding and OP GTP/GDP OP GTP Grb2 exchange phosphorylation PO PO PO PO OP of Grb2 OP OP OP involved in cell growth, PO PO PO OP PO PO PO OP differenti ati on and survi val

74 © Oxford University Press, 2013 Signal Transduction - Tyrosine Kinase Linked Receptors Example of a signalling pathway

Ras OP PO PO OP OP PO PO PO OP Gene transcription

Raf (inactive) Raf (active)

Mek (inactive) Mek (active)

Map kinase (inactive) Map kinase (active)

Transcription Transcription

factor (inactive) factor (active) 75 © Oxford University Press, 2013 Intracellular Receptors - Structure • Chemical messengers must cross cell membrane • Chemical messengers must be hydrophobic CO • Example - steroids and steroid receptors 2

Steroid binding region Zinc

DNA binding region (‘zinc fingers’)

H3N Zinc fingers con ta in CCys res idues (SH) Allow S-Zn interactions with proteins that bind to DNA and RNA 76 © Oxford University Press, 2013 Intracellular Receptors – Mechanism

Co-activator protein Receptor

DNA Messenger Receptor-ligand Dimerisation complex

Cell membrane 1. Messenger crosses membrane 2. Binds to receptor 3. Receptor dimerisation 4. Binds co-activator protein 5. Complex binds to DNA 6. Transcription switched on or off 7. Pro te in synth esi s acti vat ed or i nhibit ed

77 © Oxford University Press, 2013 Intracellular Receptors – Estrogen receptor

Binding site AF-2 regions H12 Coactivator Coactivator Estradiol

DNA

Estrogen Dimerisation & Nuclear receptor exposure of transcription AF-2 regions factor Transcription

OH OH CH3 CH3 H H

CH H H 3 How?

H H H H

O HO Testosterone Estradiol / Estrogen

78 © Oxford University Press, 2013 The oxidation of testosterone to make estradiol shows yet another way of losing a methyl group through a conjugated C=C bond. OH Methyl is removed CH3 OH and an aromatic ring CH3 is formed. CH3 H H

H H H H testosterone -estradiol O a male sex hormone HO a female sex hormone

HO H H C O H 2 CH CH2 O 2 R R R Fe +4 +3 Fe +4 Fe O 3 sp C-H oxidation O O

B H B H H H H O O B H N R H BH O O O H C H C H N R C NADH R H N R R R NAD+ O oxidation NAD+ O B H O oxidation O O H C N O R BH C

N O O N FADH FAD H B 2 H H R B H N R H H decarboxylation H dehydrogenation tautomers R H O O BH B H 79 O © Oxford University Press, 2013 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. The signal can be amplified at any 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, such as nuclear receptors and cytoplasmic 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. Nucleic receptors have DNA-binding domains containing zinc fingersand a ligand-binding domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. +2 Second messengers: Ca , lipophilics (diacylglycerol), nitric oxide and other redox signaling molecules 81 © Oxford University Press, 2013