Chaps 4 & 5: Receptors as drug targets, structure, …psbeauchamp/pdf/499_chap_4,5_81...Chaps 4 & 5: Receptors as drug targets, structure, function & signal transduction Cell signalingCell

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

Cell signaling is part of a complex system of communication that governs basic cellularCell 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.

receptorssignals

(neurotransmitters)lf

neighbor

cell 1 cell 2signals cell 3cell 2cell 1

( )

i lsynapse synapse synapse

etc.

signals

selftalk

gtalk

cell 1

cell 3cell 2

signals(hormones)receptor a receptor bsignals

(hormones)The same signal can deliver different messages to different tissues (histamine)

Important functions of receptors:1. Globular proteins (receptors) acting as a cell’s ‘letter boxes’2 Located mostly in the cell membrane

cell 4cell 5receptor dreceptor c signals

(hormones)signals

(hormones)

tissues (histamine).

© Oxford University Press, 2013 1

2. Located mostly in the cell membrane3. Receive messages from chemical messengers coming from other cells4. Transmit a message into the cell leading to a cellular effect5. Different receptors specific for different chemical messengers6. Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers

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

Autocrine signals are produced by the target cell, are secreted, and affect the target cell itself via i i ll ll l b if h h f ll h

S = signalR = receptor

receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as theemitting 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.

Rll bll S cell b

Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.

cell a S R

Rll S R

Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to reach all parts of the body (long-range allostery).

R cell bcell a Ssynapse

S R cell c S etc.

cell cR2S S

synapse synapse

© Oxford University Press, 2013

cell a SS

S S S

S S

S

S cell cR2

cell dR3cell bR1

S

S

S

S2

Central Nervous System (CNS)structureBrain and spinal cord

Peripheral Nervous System (PNS)

functionIntegrative and control centers, 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) divisionSomatic and visceral sensory nerve fibers

Motor (efferent) division (CNS)

Motor nerve fibers

Conducts impulses from the CNS to effectors (muscles and glands)

Autonomic nervous system (ANS) Somatic nervous systemSympathatic division

Conducts impulses from receptors to the CNS

Autonomic nervous system (ANS)Visceral motor (involuntary)

Somatic nervous systemSomatic motor (voluntary)

Conducts impulses from the CNS to skeletal muscles

mobilizes body systems during activity (fight or flight)

Parasympathetic divisionC

Conducts impulses from the CNS to cardiac muscles, smooth muscles, and glands


Conserves energyPromotes 'housekeeping' functions during rest

Central nervous system The central nervous system consists of the two major structures: the brain and spinal cord. The brain is encased in the k ll d t t d b th iskull, and protected by the cranium.

The spinal cord is continuous with the brain and lies on the backside to the b i d i t t d b th t bbrain, and is protected by the vertebra. The spinal cord reaches from the base of the skull, continues through or starting below the foramen magnum, and t i t hl l l ith th fi tterminates roughly level with the first or second lumbar vertebra, occupying the upper sections of the vertebral canal.

Th CNS i t t i f ti itThe CNS integrates information it receives from, and coordinates and influences the activity of, all parts of the body and it contains the majority of the

t U ll th ti dnervous system. Usually, the retina 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 napsing directl on


parts of the CNS, synapsing directly on brain tissue without intermediate ganglia..

Peripheral nervous systemThe peripheral nervous systemThe 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 mainbrain and spinal cord. Its main function is to connect the central nervous system (CNS) to the limbs and organs, essentially serving as a communication relayserving 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 bonePNS 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. Themechanical injuries. The peripheral nervous system is divided into the somatic nervous system and the autonomic nervous system


5

system

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 and defecation. Its action is described as being

Parasympathetic nervous system Sympathetic nervous system

, ( ), , g gcomplementary to that of the sympathetic nervous system, which is responsible for stimulating activities associated with the fight-or-flight response.


Somatic nervous system

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) Afferent nerves arey (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 stemand 12 are in the brain stem.

The somatic nervous system consists of three parts:

Spinal nerves: They are peripheral nerves that carry i f i i d d f hsensory information into and motor commands out of the

spinal cord.

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

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


sensory input and motor output, numbering thousands

Nerve cells make contact to muscle cells, organs and other nerve cells

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

… or synaptic button whencontact to neuron


8

axon

Synaptic button or neuromuscular junction


Cell body

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

Neurological disorders

Charcot–Marie–Tooth disease (CMT), also known as hereditary motor and sensory neuropathy (HMSN) h dit i t th d l l t h i h t(HMSN), hereditary sensorimotor neuropathy and peroneal muscular atrophy, is a heterogeneous 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

l i l di d ith 37 i 100 000 ff t d ( 1 i 2500)neurological disorders, with 37 in 100,000 affected (1 in 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 h Th di i i t d ith l d t l i th b i Thbehavioral changes. The disease process is associated with plaques and tangles in the brain. The 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

t d t th d i f l kill d t d iti d f ti hextends to the domains of language, skilled movements and recognition, and functions such as decision-making and planning become impaired.

Parkinson's disease (PD), is a degenerative disorder of the central nervous system that often i i th ff ' t kill d h P ki ' di b l t fimpairs the sufferer's motor skills and speech. Parkinson's disease belongs to 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 b the ins fficient formation and action of dopamine hich is prod ced in the dopaminergic


by the insufficient formation and action of dopamine, which is produced 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.

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 degenerationAlthough 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


12

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.

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 theSodium 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 theCa in which causes vesicles to fuse with membrane and release the neurotransmitter, starting the next impulse.

Axon

depolarizationmilli seconds

Some of these are activated by the change i b t ti l

neurotransmitters bind to receptors on the next

cell at the synapseneurotransmitters

at synapseproteins

Na Cl

C +2

KK

Axon

Cell Body

0

action potential

neurotransmittersleave cell by exocytosis

Ca+2

Na Na

action potential

in membrane potential.y p

ClNa Ca+2

Ca+2Cell Body

K

Ca+2

Na

O BHO

NH

choline retaken up by

Ion gate opens when

neurotransmitter complexes at receptor. H B

O

dendrites

O

ON

acetyl choline is an important

neurotransmitter,

diffuses across the synaptic cleft and binds to a Na+ ion-gate channel on the

ON

OH

B

O

OB

B H

choline retaken up by presynaptic neuron

BH

O H

BH

OH

O

OH


neurotransmitter,OH of a serine residue of

the receptor receptor receptorhydrolyzed back to the serine "OH"

receptor

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


14

T4


15


16

H

H

HO

H

H

H cholesterol

many steps,plus sunshine

H H

H

7-DehydrocholesterolHO

H

7-Dehydrocholesterol

HO

HH pre-vitamin D3

H

H

© Oxford University Press, 2013 17HO

vitamin D3

Hormone binding to receptor - insulin1. Insulin binds to tyrosine

kinase-linked receptor.2. Phosphorylation of receptor,

starts sequence leading to q gsynthesis of glycogen, storage of glucose (and fat too)

3. Allows influx of glucose

(Insulin protein requires post translational modification)

g4. Insulin-like molecules are

even found in the simplest unicellular eukaryotes, over 1 billion years oldy

5. Our insulin is very similar to pig and cow insulin, was used before our genes were spliced into genetically p g ymodified bacteria

© Oxford University Press, 2013 18http://www.abpischools.org.uk/page/modules/diabetes/diabetes6.cfm

(glucose transport)

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 andThere 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 asactivity, 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 controlledThe 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 leukotrienesO OO

OH

HO

OH

O

OH OH

OO

OHHO

alprostadil - inhibits blood clotting , vasodilator

O

OHthromboxane A2 - vasoconstrictor,

hypertensive, blood clotting,

lipoxin B4 - eoxin, pro-imfalmmatory (white blood cells), mast cells contributes to allergies


yp gaspirin inhibits its formation mast cells, contributes to allergies,

various cancers, Hodgkin's lymphoma

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. Steroids have twoy gprincipal 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 synthesislead to DNA activation and protein synthesis.

C Dglucocorticoids (adrenal cortex)mineralocorticoids (adrenal cortex)

A B

C mineralocorticoids (adrenal cortex)estrogens (gonads)androgens (gonads)progestins (ovaries and placenta)vitamin D (diet and sun)

Letters are used to identify the 4 rings.

vitamin D (diet and sun)...and more


20

OHCH3

H

H

H

H

H

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 i l d t t h ti l f ti i ll It i 2 t

HO

levels in men are lower compared to women, estrogens have essential functions in men as well. It is 2 stepsaway from progesterone.

OHCH3

H

H

H

H

H

CH3

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 f l d i i h h i d ll i d

HH

O


development of male reproductive tissues such as the testis and prostate as well as promoting secondarysexual 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.

CH3 H

CH3

HCH3

CH3

CH3

HH3C

H

H3CH

CH3 H

CH3C

H

OCH3

V22, p. 703, p 1263

about 19 steps

? steps

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

lanosterol - the compound from which all steroids are derived.

HO cholesterol - essential component of cell walls, precursor of steroid hormones, bile acids and vit D

HO

H H

H3C CH3H

CH3

HO

H H

pregnenolone - neuroactive steroid

CH3C

OH

OCH3 CH3

C

OH

OCH3 CH3

C

H

OCH3

NAD+ and isomerization

sp3 oxidation

some operating at the nanomolar level.

O OH

HO

CH3

H

H

H

OH

17-hydroxypregnenolone - higher levels are found at end of puberty and in pregnacy.

17-hydroxyprogesterone - sometimes given to prevent premature birth

O

CH3

H

H

H

O

CH3

H

H

H

progesterone - involved in menstrual cycle and pregnacy

O OH

NAD+ and isomerization sp3 oxidation

sp3 oxidationsp3 oxidation? steps

squalene oxide

O

O

CH3

H

H

H

CH3C

OH

CH2

O

CH3

H

H

H

CH3C

H

CH2

11-deoxycorticosteroneintermediate to aldosterone

CH3

H

H

H

HO

O

CH3

d h d i d t b d

? steps

squalene

Statins drugs work here.

O OO

CH3

H

H

H

CH3OH

H

11-deoxycortisol (cortodoxone)glucocorticoid (similar to cortosol)

CH3 H

CH3C

OHHO

OCH2

OH

intermediate to aldosterone

CH3

H

H

H

CH3C

H

OCH2

OH

HO

dehydroisoandrosterone - most abundantcirculating steroid, many uses

reduced C=C is

sp3 oxidation

sp3 oxidationNADH

many steps

O

dimethylallyl phosphate

isopentenyl phosphate

P

OO

O P

OO

testosterone - produced in all vertebrates, 20x more in males, male sex characteristics.

CH3

H

OHthree well known members of the estrogen family

CH3

H

OH

O

H H

C C

OHHO

OCH2

OH

O H

cortisol - glucocorticoid, increases blood sugar, decreases immune system (hydrocortisone)

O

H HO

H H

corticosterone - stress hormone, immune system

5 categories of steroid hormones1. progestins - overies and placenta- mediate menstrual cycle and maintains pregnacy2. glococorticoids - adrenal cortex - affect

metabolism in diverse ways decrease

CH3

H

O

OH

dihydrotestosterone(maybe with FADH2)(more potent by 3x)

sp3 oxidationand

NAD+ oxidationseereactions

below

O

SCoAacetyl CoA

© Oxford University Press, 2013 22-estradiol - prominant estrogen in reproductive years, more potent than estriol (10x) and estrone (80x)

estrone - known female carcinogen, causes breast tenderness,in men causes anorexia

HH

HO

H

H

H

HO O

CH3

H

H

H

aldosterone - Na+,K+ balance, blood pressure reg.

metabolism in diverse ways, decrease inflamation,incease resistance to stress3. mineralocorticoids - adrenal cortex - maintain salt and water balance4. androgens - gonads - maturation and function of secondary sex organs, particularly in males, male sexual differentiation5. estrogens - gonads - maturation and function of secondary sex organs, particularly in females

H

H

H

HO

estriol - higher levels in pregnancy, marker of fetal health

OH

sp3 oxidationNAD+

oxidation

Amino acid derivatives (or from amino acids) – thyroxine, histamine, catacholamines (epinephrine, norepinephrine) oxytocin, cytokines, glutamate, glycine, gama aminobutyric acid, acetylcholine (neurotransmitters and hormones)

OO HOO

O

NH3

H2N

epinephrine

O

O

HO

HO

NH3HO

HO

O

NH3glutamate norepinephrineadrenaline

O

HOHO

O

HO

I

I

OH3N

glycine

O

-aminobutyric acid

H3N O

NH3

th i (T4)

OI

NO

O

thyroxine (T4) I

nitrosyl di l

NON

HN

NH3NH3

HO

HO

acetylcholine

SS

CysTyr

oxytocin - 8 amino acid peptide

radical(nitric oxide)histaminedopamine

© Oxford University Press, 2013 23Gly Leu Pro Cys

S

Asn Ile

oxytocin 8 amino acid peptide

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 on a target cell, such as a muscle cell or another neuron. Usually short lived p g , yand 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 on target cells throughout the body

Nerve 1

p g g y

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

Synaptic button =, connections to neurons,

l h li

Blood supply e g

acetylcholine

Neuromuscular end plates = connections to muscles,

adrenoline

receptors

Nerve 2Hormone

Blood supply, e.g.insulin – absorb glucoseGlucagon – release glucose response

Cell


Neurotransmitters

24

secondarymessenger

Mechanism Structure and function of receptors

Messenger Induced fit

Messenger

Messenger

CellMembrane

Cell

Receptor

messageCell

Receptor

Messenger

MCell

Receptor

messageMessage

Receptors contain a binding site (hydrophobic hollow or cleft on the receptor protein) that is recognised by the chemical messenger

ion - ionH bondsBinding of the messenger involves intermolecular bonds with amino acids

Binding results in an induced fit of the receptor protein and the messenger (alters shape)

H bondsdipole - dipoledispersion forces

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

Binding interactions

• IonicIonic• H-bonding• van der Waals

Induced fit - Binding site alters shape to maximise intermolecularbonding

PhePhe

SerO

H

Asp

CO2 Induced Fit

Ser

OH

Asp

CO2

Asp Fit Asp


Intermolecular bonds not optimum length for maximum binding strength

Intermolecular bond lengths optimised, change of shape causes a change in function (on or off) 26

Overall Process of Receptor/Messenger Interaction

M

M MM = messengerR = receptor

EER R EER

Bi di i i b h h ld h ffi i l l

Signal transduction

Binding interactions must be strong enough to hold the messenger sufficiently long for signal transduction to take place

Interactions must be weak enough to allow the messenger to departInteractions 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

Receptor Superfamilies RESPONSETIME

•ION CHANNEL RECEPTORS

•G PROTEIN COUPLED RECEPTORS

msecs

d•G-PROTEIN COUPLED RECEPTORS

•KINASE LINKED RECEPTORS

MEMBRANE BOUND

seconds

minutes

•INTRACELLULAR RECEPTORS


28

Ion Channel Receptors

General principlesGeneral 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+)• 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 cell29

Ion Channel Receptors - General principleshydrophilic

tunnel

Cellmembrane

hydrophobicexterior

IONCHANNEL

( )

RECEPTORBINDING

IONCHANNEL

(closed)

Extra cellularExtra cellularMESSENGER

Induced fit and opening

of ion channel

(open)

Cellmembrane

MESSENGER

Ionchannel

Ionchannel

Cellmembrane

SITE(closed)

Lock Gate Ion

channelIon

channelCell

membraneCell

membrane


CellCell

30

Ion Channel Receptors - Gating

MessengerReceptor Binding site

InducedfitCell

membrane Cellfit

‘Gating’(ion channel

opens)

membrane Cellmembrane

Five glycoprotein subunitstraversing cell membrane

Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatoryAnionic ion channels for Cl- (e.g. GABAA) = inhibitory


31

Structure of protein subunits (4-TM receptor subunits)

Ion Channel Receptors

Structure of protein subunits (4 TM receptor subunits)

Extracellular loopExtracellular loop

Neurotransmitter binding regionNeurotransmitter binding region

H2N

CO HCellCellmembranemembrane

one of

these CO2H

CellCellTM1 TM2 TM4TM3

membranemembrane these

IntracellularIntracellularlooploop

Variable loopVariable loop

membranemembraneTM1 TM2 TM4TM3

pp

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


Ion Channel Receptors - Detailed Structure of Ion Channel

Proteinsubunits

TM4

p

subunits

TM4TM4

TM3

TM3 TM2

TM2TM2

TM1

TM1

Transmembrane

TM4TM4

TM3

TM3

TM2

TM2TM2

TM2

TM1

TM1

TransmembraneregionsTM3

TM3 TM2TM2 TM1

TM1TM4 TM4

Note: TM2 of each protein subunit ‘lines’ the central pore(TM2 is more polar, perhaps bonded to very polar carbohydrates, TM1, TM3 and TM4 are more hydrophobic and face towards cell membrane.)


3 d e o e yd op ob c d ce ow ds ce e b e.)

33

Ion Channel Receptors - Gating Ion flow

Cellmembrane

TM2 TM2

Transverse view

TM2TM2

TM2

TM2

TM2 Transverse viewTM2

TM2

TM2TM2

•• Chemical messenger binds to receptor binding site

ClosedOpen

TM2

• 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


• Ion concentration within cell alters • Leads to variation in cell chemistry

Ion Channel Receptors – Nicotinic receptor

Bi di

Ion channel

Bindingsites

Cellmembranee

Two ligand binding sitesmainly on -subunits

x subunits mainly on subunits

O

N

H3CCH3

N OCH3

N

CH3

H3C

H CH3C O CH3

acetylcholineneurotransmitter

nicotineprotonated at body pH

N

CH3H

OH3C

OHMuscarine


p y p OHMuscarineMuscarinic agonists activate muscarinic receptors while nicotinic agonists activate nicotine receptors. Both are direct-acting cholinomimetics that mimic acetyl choline and act on the CNS.

Ion Channel Receptors – Glycine receptorp y pBindingBindingsitessites

Ion channelIon channel

CellCellmembranemembrane

Three ligand binding sitesThree ligand binding siteson on --subunitssubunitsxxxx subunitssubunits

The glycine receptor, or GlyR, is the receptor for the amino acid neurotransmitter O

glycine

glycine. GlyR is an ionotropic receptor that produces its effects through chloridecurrent. 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.

H3NO


brainstem.

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

messenger

i d d

G protein

inducedfit

closed open

G-protein split into α and β/ subunits


α and β/ subunits

37

G-Protein Coupled Receptors – General principles• G-Protein subunit activates membrane bound enzyme• Binds to allosteric binding site• Induced fit results in opening of active site

p p p p

• Induced fit results in opening of active site• Intracellular reaction catalysed (makes cyclic AMP, PIP2, etc.)

Enzyme Enzyme

active site active site( )

α α

(closed) (open)

I t ll l


Intracellular reaction

38

G P t i C l d R t St tSingle protein with 7 transmembrane regions (7-TM receptor)

Extracellular

G-Protein Coupled Receptors - Structure

Extracellularloops

N -Terminal chain

NH3

extracellular

TransmembranehelixVII VI V IV III II IMembrane

G-Proteinbinding region

VariableO2C

cytoplasm

C -Terminal chainVariableintracellular loop Intracellular loops

G protein splits into an subunit and an / subunit.


39

varies depending on receptor typeG-Protein Coupled Receptors – Ligand binding sitevaries depending on receptor type

Ligand

A) Monoamines: pocket in TM helicesB) Peptide hormones: top of TM helices + extracellular loops + N-terminal chainC) H t ll l l + N t i l h i

B DCA

C) Hormones: extracellular loops + N-terminal chainD) 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, testosterone, estrogen, oxytocin, epinephrine


y , , g , y , p p• Glutamate, DOPA, glycine, acetylcholine, Ca++

SIGNAL TRANSDUCTION

neurotransmitter or hormone

Overview signal binds at receptor site of 7 TM receptor There are many different types of G

proteins with about 20 different types of α subunit proteins.

G protein subunits

, , subunits

G protein-coupled receptors (7 TM receptors) imbedded in membranes

subunit splits offand binds to another protein to start signal cascade inside the cell

continuing

© Oxford University Press, 2013 41protein

proteincontinuingreactions inside cell

variousactions

inside cell

Signal Transduction involving Gs-Proteins provide one step in signalDRUG TARGETS SIGNAL TRANSDUCTION

transduction pathway.

GGSS--ProteinProtein - membrane bound protein of 3 subunits ()b it h bi di it f GDP- S subunit has binding site for GDP

-GDP bound non covalently

GDPInteraction of receptor with Gs-protein

ß

GTP binds

I d d fit

Fragmentationand release ß

ß

Binding site recognises GTPGTP = guanine triphosphate

Induced fit G-protein alters shapeComplex destabilised

• Process repeated for as long as ligand bound to receptor


• 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

b itb itGTPGTPSignal Transduction involving Gs-Proteins

Interaction of s with adenylate cyclase (inside cell) ss--subunitsubunit

Adenylate cyclaseAdenylate cyclase

GTPGTPGDPGDP

Binding sitefor s subunit

Binding

GTP hydrolysed to GDP catalysed by subunitBinding

Inducedfit

P

by s subunit

cyclic AMPcyclic AMPATPATP cyclic AMPcyclic AMPATPATPαs αs

Active site(closed)

Active site (open)

Subunit changes shape

Active site(closed)

cyclic AMPcyclic AMPATPATP cyclic AMPcyclic AMPATPATP

Protein

Activation

s Subunit changes shapeWeaker binding to enzymeDeparture of subunitEnzyme reverts to inactivestate

Kinase A

Enzyme(i ti )

Enzyme( ti )

PP


s Subunit recombines with dimer to reform Gs protein

state

43

(inactive)

Enzyme-catalysedreaction

(active)

P t i ki A 4 t i b it

Signal Transduction involving Gs-ProteinsInteraction of cyclic AMP with protein kinase A (PKA)Protein kinase A - 4 protein subunits

- 2 regulatory subunits (R) and 2 catalytic subunits (C)cAMPcAMP

CC catalytic subunit releasedcatalytic subunit released

CC

CCRR

RR

cAMPcAMPRR

RR

• Cyclic AMP binds to PKA• Induced fit destabilises complex• Catalytic units released and activated

CCcAMP cAMP binding binding sitessites

catalytic subunit releasedCC

• Catalytic units released and activated

C

Protein Protein

PPhosphorylation of other proteins and enzymesSignal continued by phosphorylated proteinsFurther signal amplification


+ ATP + ADP

Signal Transduction involving Gs-ProteinsGlycogen Metabolism - triggered by adrenaline in liver cells

Ad liAdrenaline

Adrenoreceptor

s

adenylate

s

signalreceived

cAMP

Protein kinase AGlycogen

-Adrenoreceptor ycyclase

Protein kinase A

Catalyticsubunit ofPKA

C

Inhibitor (inactive)

Inhibitor-P Phosphatase

Glycogensynthase(active)

Glycogen

Phosphorylasekinase (inactive)

Phosphorylasekinase P (active)

Inhibitor-P(active)

Phosphatase(inhibited)

Glycogensynthase-P(inactive)

kinase (inactive) kinase P (active)

Phosphorylase b(inactive)

Phosphorylase b(active)

© Oxford University Press, 2013 45Glycogen Glucose-1-phosphate

Signal Transduction involving Gs-Proteins

Interaction of s with adenylate cyclase converts ATP to cAMP

NH2ATP NH

N

NN

N

OOPO

O

P

O

OP

O

O

adenosine triphosphateBB H

N

NN

N

NH2

OP

O

OP

O

O

cyclic AMP

O

H

O

HO

HH

HH

OO O

H B

enzymecontrol O

H

HH

H

OPOPOP

O O

O

OH

O

diphosphate(pyrophosphate) BH

• Several 100 ATP molecules converted before s-GTP deactivated• Represents another signal amplification• Represents another signal amplification• Cyclic AMP becomes next messenger (secondary messenger)• Cyclic AMP enters cell cytoplasm with message (possible further amplification)


Glycogen Metabolism triggered by adrenaline in liver cells

Signal Transduction involving Gs-Proteins

Coordinated effect: activation of glycogen metabolism ( glucose glycolysis) energy)inhibition of glycogen synthesis (no gluconeogenesis)

Glycogen Metabolism - triggered by adrenaline in liver cells

Adrenaline has different effects on different cellse.g. activates fat metabolism in fat cells ( acetyl CoA glucose = slower)

Drugs interacting with cyclic AMP signal transductionDrugs interacting with cyclic AMP signal transduction

Theophylline and caffeine -inhibit phosphodiesterases

Cholera toxin causes constant activation of cyclic AMP leading to diahorrea

-inhibit phosphodiesterases -phosphodiesterases responsible for metabolizing cyclic AMP-cyclic AMP activity is prolonged (keeps you wired)

O CH3O H

N

N N

N

O

H3C

3

N

N N

N

O

H3C

H


NO

CH3

Caffeine

N NO

CH3

Theophylline

Interaction of cyclic AMP with protein kinase A (PKA)Signal Transduction involving Gs-Proteins

Protein kinase A is a serine-threonine kinaseActivated by cyclic AMPCatalyses phosphorylation of serine and threonine residues on proteinsubstrates

Phosphate unit provided by ATPO O

HN

NH

ProteinProtein

O

Proteinkinase A

HN

NH

ProteinProtein

OP

O

O

O

O ATPO ADP

ADPB P

phosphorylated serine or threonine residue

serine or threonineamino acid

OO

O

OATPH

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 Up to 30%


contains about 500 protein kinase genes 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.

• Rhodopsin = visual receptor

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• Implications for drug selectivity depending on receptorsimilarity – consequence of evolution

• Membrane bound receptors difficult to crystallise• X Ray structure of bacteriorhodopsin solved bacterial• X-Ray structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsin

• Bacteriorhodopsin structure used as ‘template’ for othertreceptors

• Construct model receptors based on template and aminoacid sequenceL d t d l bi di it f d d i• Leads to model binding sites for drug design

• Crystal structures for rhodopsin and 2-adrenergicreceptors now solved - better templates


CC = 2pa - 2pb = antibonding MO pi-startib d2p 2 C Chigher

nodeEthene has a pi bond. Pi bonds are second and third bonds and are generally weaker. MO diagram of a pi bond.

antibond2pa

C C

2pbC C

potential energy

higher,less stable

LUMO

CC = 2pa + 2pb = bonding MO pi bondC C lower,more stable

HOMO

HOMO = highest occupied molecular orbitalLUMO = lowest unoccupied molecular orbital

four C empty, but available to accept electrons

CC = sp2 - sp2b = antibonding MO

Ethene MO diagram

H H

HOMO = highest occupied molecular orbital

CC sp a sp b antibonding MO

CC = p2a - p2

b = antibonding MOC

CH H

H Henergy of startingatomic orbitals

LUMO

2C = 8 e's4H = 4 e's

total bonding e's = 12 e's CC = sp2a + sp2

b = bonding MO

CC = p2a + p2

b = bonding MO 63 kcal/mol

83 kcal/mol

146 kcal/molHOMO


four C full, with two electron each

bond order = (12) - (0) 2 = 6 bonds

C C

HH

H

node nodenodeMore nodes is less stable = no

electron density between adjacent atoms.buta-1,3-diene - conjugated pi bonds

node

C C H

HH node

4 = 1 - 2

4

nodenode

C C C C

C C higher,less stable

C C1

23 C C C CLUMO LUMO

potential energy 2 = 1 - 2 = HOMO

3 = 1 + 2 = LUMO

nodeE is larger

E is smallerso is longer

C Cl C C

2 C C C CC

C C lower,more stable

C C

1 = 1 + 2

12

1 C C C CCEnergy gap between HOMO and LUMO orbitals in pi bond versus diene.

HOMO HOMO

© Oxford University Press, 2013 51E = h = hc(1/)h = Plank's constant = 6.62x10-34 j-secc = 3.0x108 m/sec

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 the energy.

systems max (wave length measure of HOMO/LUMO energy gap

H2C CH2 174 nmlarger E (HOMO/LUMO energy gap)

The HOMO/LUMO energy gap can be measured with light (frequency = or wave length = )

217 nm

250 nm

280 nm

smaller , indicates a larger E

E = (hc) 1

*

*

310 nm

340 nm

370 nmsmaller E (HOMO/LUMO energy gap)

larger , indicates a smaller E

*

*

*

400 nm

smaller E (HOMO/LUMO energy gap)

* = estimated value

*

100 nm 200 nm 300 nm 400 nm 500 nm 600 nm 700 nm 800 nm 900 nm

1 2 3 4 5 6 7 8

ultraviolet light

visible light

infrared light

Increasing EnergyIR is lower energy electromagnetic

radiation that we sense as heatUV is higher energy electromagnetic

radiation that can damage chemical bonds

V I B G Y O R

-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 (?) =

E

LUMO

transitionE

100 nm 200 nm 300 nm 400 nm 500 nm 600 nm 700 nm 800 nm 900 nm

Increasing Energy

eyes detect electromagnetic radiation (light) in this regiontransitions in visible

higher energy lower energy

Ultra Violet InfraredV I B G Y O R

HOMO

transition in UV

radiation (light) in this regiontransitions in visible

-carrotene

oxidation

OO

hydration

OH

id ti OHoxidation

OH

vitamin Aoxidation

H

recycles through vitamin A via1. hydrolysis2. reduction

rhodopsin

O

11-trans-retinalisomerization

11-cis-retinal

O P S I N

protein

O P S I N

DOHR

Binding is weak when trans configuration.

OH

binds to opsin protein

O P S I NH3N

Absorption of photon (h) causes isomerization of system to the more stable trans configuration.

H

N

HOHR D

Rhodopsin absorbs visible light (depends

Aldehyde and 1o amine bind together in an imine linkage with loss of water.

© Oxford University Press, 2013 53NH

H

p g ( pon the protein charge and twist inconformation), 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".

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.

Binding is strong when cis configuration.

What would happen if one complementary

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 / )

How does vision work?

E = h

proteinA

proteinB

proteinC

R = redO = orangeY = yellowG = greenB = blueI indigo

of these was defective? complementarycolor wheel

red

orange

yellowlt blue

dk blue

violetA B C

Rhodopsin complex imbedded in cell membrane has retinal bound perpendicular to 7 transmembrane protein helicies. Isomerization triggers cell action potential (polarization) and Na+ flows in and K+At a synapse, neurotransmitters are released from vesicles,

I = indigoV = violet

yel-grngreen

lt blue

= 400 nm = 700 nmV I B G Y O R

AC = acetylcholine used in peripheral and

l

O

N

triggers cell action potential (polarization) and Na flows in and Kflows out (1 millisec). In some cells this is followed by Ca+2 ions efflux (100 millisec).

Na+

y p , ,triggering the next neuron to fire and tranmit the signal to the appropriate parts of the brain

ion channelsACAC

AC

cell membranecell membrane (lipid bilayer)

C

OO

GLU = glutamate / glutamic acid

central nervous systemsO

N

GLU

AC

vesicles with neurotransmittersfuse with cell wall and release

1000s of molecules = exocytosisRhodopsin

complex

HO CNH3

CO

NH3

OGLU = glutamate / glutamic acid

important neurotranmitter in the brain and spinal cord

NH3HODPA = dopamine used in several parts of the brain, cocaine and

methamphetamine act on dopamine

GLUGLU

GLU

HO2C


HO

methamphetamine act on dopaminereceptors, Parkinson's is caused by loss of dopamine secretion K+

Ca+2 ion channelsDPADPA

DPA

cell membrane

G-Protein Coupled Receptors – bacteriorhodopsin / rhodopsin family

Common ancestor divergent evolution

Monoamines

betaalphamuscarinic Opsins,

Rhodopsins

Receptortypes

Endothelins BradykininAngiotensinInterleukin-8

Tachkinins

Muscarinic Histamine -Adrenergic Dopaminergic -Adrenergic

Receptorsubtypes24 5 3 1 D4H2 2A 2B 2C1 13H1 D3 D2 D1A D5D1B2

convergent evolution convergent evolution


55

Reflects differences in receptors which recognise the same ligandG-Protein Coupled Receptors – Receptor types and subtypesReflects differences in receptors which recognise the same ligand

ReceptorReceptor TypesTypes SubtypesSubtypes

Al h ( )HOH

Alpha ()Beta ()

1, 2A, 2B, 2C

1, 2, 3

AdrenergicNCH3

HO

HO

adrenoline

O CH3

Cholenergic NicotinicMuscarinic

ON

CH3

CH3acetylcholine

•Receptor types and subtypes are not equally distributedamongst tissues

• Target selectivity leads to tissue selectivityTarget selectivity leads to tissue selectivity

Heart muscle 1 adrenergic receptorsFat cells 3 adrenergic receptors Bronchial muscle & adrenergic receptors


Bronchial muscle 1& 2 adrenergic receptorsGI-tract 1 2 & 2 adrenergic receptors

Signal Transduction involving Gi-Proteins (i = inhibition)

• Bind to different receptors from those used by Gs proteins

• Mechanism of activation is identical• 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 ( or )• Overall effect depends on dominant alpha subunit (s or i)

• Dominant alpha subunit depends on receptors activated


Phosphorylation Reactions

• Prevalent in activation and deactivation of enzymes• Phosphorylation radically alters intramolecular binding

R lt i lt d f ti• Results in altered conformations

NH3NH3NH3

O

NH3

PO

OO

3

H

O PO

O

O

might turn on, might turn off

OOOO

Active siteActive siteclosedclosed

Active siteActive siteopenopen


Phosphorylation Reactions

• Prevalent in activation and deactivation of enzymes• Phosphorylation radically alters intramolecular binding

R lt i lt d f ti• Results in altered conformations

CO2 CO2 CO2

OH PO3-2

O PO

O

might turn on, might turn off

O





Signal Transduction involving Gq ProteinsInteraction 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 subunitq• 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 siteActive site(closed)(closed)

PLCPLC

Active siteActive site(open)(open)

PLCPLC

PLCPLC PIP2

DG

PLCPLC PLCPLC PLCPLC 2

Active siteActive site

IP3

GTP hydrolysisGTP hydrolysis qq departsdeparts (closed)(closed)

EnzymeEnzyme

PLCPLCPLCPLC PIPPIP22

DG

IP3PhosphatePhosphate


Binding Binding weakenedweakened

EnzymeEnzymedeactivateddeactivated

IP3PhosphatePhosphate

60

Signal Transduction involving Gq ProteinsInteraction with phospholipase C (PLC)

O O

C O

R

CO

R

BH

H OHO

O

H2CC

HH2C

PP

OO OH

H

B O

OH O C O

R

CO

RP

O

O

HO

O

OH

OH

OP

PLC

phospholipase C

OOH

O

CH2CH

H2C

O O OH

Phosphatidylinositol diphosphate Inositol triphosphate Diacylglycerol

OP

PIP2

IP3 DGinositol triphosphate

phosphatidylinositol diphosphate

p p p OP

diacylglycerol

Phosphatidylinositol diphosphate(integral part of cell membrane)

Inositol triphosphate(polar and moves into cell cytoplasm)

Diacylglycerol(remains in membrane)


P = PO32-R= long chain hydrocarbons

61

Action of diacylglycerolSignal Transduction involving Gq ProteinsAction of diacylglycerol

• Activates protein kinase C (PKC)• PKC moves from cytoplasm to membrane

Ph h l t S & Th id f t i b t t• Phosphorylates Ser & Thr residues of protein substrates• Activates enzymes to catalyse intracellular reactions• Linked to inflammation, tumour propagation, smooth muscle activity etc

DGBindingBinding

Cell membrane

DG

PKC

site for DGsite for DGPKC


PKCDG

EnzymeEnzyme(inactive)(inactive)

EnzymeEnzyme(active)(active)

Enzyme catalysed

Cytoplasm

PKC movesto membrane

Cytoplasm

DG binds toDG binding site

Cytoplasm

Induced fitopens active site

Enzyme-catalysed reaction


g p

62

Resynthesis of PIP phosphatidylinositol diphosphate

Signal Transduction involving Gq Proteins

Resynthesis of PIP2 – phosphatidylinositol diphosphate

IP3 IP2 IP inositol PI PIP3 PIP2phosphatase phosphatase phosphatase PI synthase PI-4-kinase PI-4-P-5-kinase

inhibition

Li salts for manic depressive illness

OO IP3 HO HH HOO

O

R

OR

PIP2

OHO

O H

P

O

O

O

P

O

O

O

IP3

OH

H

HO

H

HO HO

O H

P

O

O

O

P

O

O

O 2

H

OH

OH

H

HO

PO

HO HH HO

inositol

O

H

OH

HO

H

HO

O

O

O

O

R

OR

P

OO

OHP

O

OOOH

O

DG = diacylglycerol


63

Signal Transduction involving Gq ProteinsAction of diacylglycerol

O

Drugs inhibiting Protein Kinase C (PKC) - potential anti cancer agents

O HO

MeHOMe

Me

O

OO O

H

O

HO

OH

Me

Me

O

O

H

HH

O

O

MeH

OHO

O

Me

B t ti (f )


64

Bryostatin (from sea moss)

Text, Fig 21.70 p. 566

Action of inositol triphosphateSignal Transduction involving Gq Proteins

•IP is hydrophilic and enters the cell cytoplasm•IP3 is hydrophilic and enters the cell cytoplasm• Mobilizes Ca2+ release in cells by opening Ca2+ ion channels• Ca2+ activates protein kinases• Protein kinases activate intracellular enzymes• Cell chemistry is altered leading to a biological effect

Calmodulin = calcium modulated protein, interacts with kinases and phosphatases

Cell membrane

IP3 CytoplasmCytoplasm

Calciumstores CaCa++++

CalmodulinCalmodulin CaCa++++

ActivationActivationActivationActivation

Proteinkinase

Proteinkinase

Enzyme(active)

Enzyme(inactive)

PP

Enzyme(inactive)inactive)

Enzyme(active)

PP


EnzymeEnzyme--catalysedcatalysedreactionreaction

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

EnzymeEnzyme--catalysedcatalysedreactionreaction

Tyrosine Kinase Linked Receptors•Bifunctional receptor / enzyme

A i d b h• Activated 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• Protein changes shape and opens intracellular active site• Reaction catalysed within cell• Overexpression related to several cancers

messenger

i d d

messenger

inducedfit

closed active site open closed

© Oxford University Press, 2013intracellular reaction 66

Tyrosine Kinase Linked Receptors - Structure

ExtracellularExtracellularNN--terminalterminal

Tyrosine Kinase Linked Receptors Structure

N H 3LigandLigand binding regionbinding region

NN terminalterminalchainchain

H d hiliH d hili

Cell membrane

Hydrophilic Hydrophilic transmembranetransmembraneregion (region (--helix)helix)

Catalytic binding region Catalytic binding region (closed in resting state)(closed in resting state)

IntracellularIntracellularCC terminalterminalCC--terminalterminalchain in chain in cytosolcytosol

C O 2


67

Tyrosine kinase

Tyrosine Kinase Linked Receptors reaction catalyzed by tyrosine kinaseTyrosine Kinase Linked Receptors – reaction catalyzed by tyrosine kinase

H

OH

O

HN

NH

ProteinProtein Tyrosine

kinaseMg+2

HN

NH

ProteinProtein

P

O

O

O

O ATP O ADP

OADP

H

B

O

PPhosphorylated tyrosine residue

Tyrosineamino acid

O

O

O

ATP

B tyrosine residue O


68

Tyrosine Kinase Linked Receptors – Epidermal growth factor receptor (EGF- R)

Ligand binding

EGFEGF

Ligand binding & dimerisation

OHOHOH

HO

Phosphorylation

ATP ADPOP

OPOPPO

Cellmembrane

Binding site for EGF

Induced fitopens tyrosine kinase active sites

OHOH ATP ADP OPOP

Inactive EGF-R monomers

Binding site for EGFEGF - protein hormone - bivalent ligandActive 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

l i i i f i lli i d


69

• Results in activation of signalling proteins and enzymes• Message carried into cell

Tyrosine Kinase Linked Receptors Insulin receptor (tetrameric complex)

Insulin

Tyrosine Kinase Linked Receptors Insulin receptor (tetrameric complex)

Phosphorylation

OP

p y

ATPADP OP

OPPOOH

OHOHHO

Cellmembrane

Insulin binding siteKinase active site

Kinase active siteopened by induced fit


70

Tyrosine Kinase Linked Receptors - Growth hormone receptorTetrameric complex constructed in presence of growth hormone

GH

GH binding&

dimerisation

ATP

Activation and phosphorylation

Binding of kinases

Kinases

GH receptors(no kinase activity)

OPOPOP

PO

ATPADP

OHOHOH

HO

Growth hormone binding site

Kinase active siteopened by induced fitOH

OHOH

HO

Growth hormone binding siteKinase active site


71

Si lli th

Signal Transduction - Tyrosine Kinase Linked Receptors

Signalling pathways

1-TM Receptors

Tyrosine kinaseinherent or associated Guanylate cyclase

Signalling proteins cGMP

PLC IP3 GAP Grb2 Others O

kinase

PIP3IP3 DG

N

NN

N

OOPO

O

cyclic GMP

NH2

H


Ca++ PKC 72

O

H

HH

HO

H

Signal Transduction - Tyrosine Kinase Linked ReceptorsExample of a signalling pathway

Growthfactor

p g g p y

factor

1) Binding ofgrowth factor Dimerisation Phosphorylation

OHHO

HO OH

2) Conformationalchange

OHHO

HO OH

OHHO

HO OHOH

HO

OH OHOP

PO

POOP

PO

PO OPPO

OH

Grb2Binding

Grb2OP

PO

PO

OPPO

PO

OPBinding and phosphorylation

of Grb2

Grb2 Ras and

GTP/GDPexchange

OPPO

POOPPO

PO

OPGDPGTP

Ras

involved in cell growth, diff ti ti d i l


PO PO OPPO PO PO PO OP

73

differentiation and survival

Signal Transduction - Tyrosine Kinase Linked ReceptorsExample of a signalling pathway

Growthfactor

p g g p y

factor

1) Binding ofgrowth factor Dimerisation Phosphorylation

OHHO

HO OH

2) Conformationalchange

OHHO

HO OH

OHHO

HO OHOH

HO

OH OHOP

PO

POOP

PO

PO OPPO

OH

Grb2Binding

Grb2OP

PO

PO

OPPO

PO

OPBinding and phosphorylation

of Grb2

Grb2 Ras and

GTP/GDPexchange

OPPO

POOPPO

PO

OPGDPGTP

Ras

involved in cell growth, diff ti ti d i l


PO PO OPPO PO PO PO OP

74

differentiation and survival

Signal Transduction - Tyrosine Kinase Linked ReceptorsExample of a signalling pathwayExample of a signalling pathway

Gene transcriptionOPPO

POOPPO

PO OPPO

OPRas

Raf (inactive) Raf (active)

Mek (inactive) Mek (active)

Map kinase (inactive) Map kinase (active)


Transcription factor (inactive)

Transcription factor (active) 75

Intracellular Receptors - Structure• Chemical messengers must cross cell membrane

CO2

Chemical messengers must cross cell membrane• Chemical messengers must be hydrophobic • Example - steroids and steroid receptors

Steroidbinding region

Zincbinding region

DNA binding region(‘zinc fingers’)

Zi fi t i C id (SH)

H3N


Zinc fingers contain Cys residues (SH)Allow S-Zn interactions with proteins that bind to DNA and RNA

76

Intracellular Receptors – MechanismCo-activator

proteinReceptor

protein

MessengerReceptor-ligand

complexDimerisation

DNA

Cellmembrane 1. Messenger crosses membrane

2. Binds to receptor3. Receptor dimerisationp4. Binds co-activator protein5. Complex binds to DNA6. Transcription switched on or off7 P t i th i ti t d i hibit d


7. Protein synthesis activated or inhibited

77

Intracellular Receptors – Estrogen receptorIntracellular Receptors Estrogen receptor

H12

Binding site AF-2

regions CoactivatorCoactivator CoactivatorCoactivator

Estradiol

H12 g CoactivatorCoactivator CoactivatorCoactivator

DNADNA

Estrogenreceptor

Dimerisation &exposure of AF-2 regions

Nucleartranscription

factor Transcription

OHCH3

H

H

OHCH3

H

H

CH3

HH

HO

HH

O

3 How?


Estradiol / EstrogenTestosterone

OHCH3

OHCH3Methyl is removed

and an aromatic ring is formed

The oxidation of testosterone to make estradiol shows yet another way oflosing a methyl group through a conjugated C=C bond.

H

H

H

HO-estradiol

a female sex hormone

H

H

H

Otestosterone

a male sex hormone

CH3

is formed.

O

H2C

R

H

Fe

OH

CH2

R

O

CH2

R

HO

Fe

O

+4 Fe+3

3

+4O

O O

N R

BO

H HB

B HC

R

OH

H

H

C

OH C

OO

H

HH

B

N R

sp3 C-H oxidation

N RH

H

NADH

O

O

R

O

R

C

OO

HB

ON

H

NAD+oxidation NAD+

oxidation

O

R

BH

C

ON

NH H

B H

R

H

N

N

HHBR

FADFADH2

d b l ti

© Oxford University Press, 2013 79O

R

H

H H

BO

RH

B HO

Htautomers

decarboxylationdehydrogenation

Overview of signal transduction pathways


80

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 stepexpression, 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 anTyrosine kinase receptors are transmembrane proteins with an intracellular kinase domain and anextracellular 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 proteinsIntracellular receptors, such as nuclear receptors and cytoplasmic receptors, are soluble proteinslocalized 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

i N l i t h DNA bi di d i t i i i fi d li d bi di


expression. Nucleic receptors have DNA-binding domains containing zinc fingers and a ligand-bindingdomain; the zinc fingers stabilize DNA binding by holding its phosphate backbone.

Second messengers: Ca+2, lipophilics (diacylglycerol), nitric oxide and other redox signaling molecules

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Chaps 4 & 5: Receptors as drug targets, structure, …psbeauchamp/pdf/499_chap_4,5_81...Chaps 4 & 5: Receptors as drug targets, structure, function & signal transduction Cell signalingCell