signal transduction

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No man is an Iland, intire of it selfe; every man is a peece of the Continent, a part of the maine; …

any mans death diminishes me, because I am involved in Mankinde;

And therefore never send to know for whom the bell tolls; It tolls for thee.

John Donne; Meditation XVII

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SIGNAL TRANSDUCTION• Even though the efficiency of the tissues is raised

through specialization, there should be coordination of activities

• Cells communicate and adapt to changes to their environment using various signals

• Extracellular signaling molecules or ligands are synthesized and released by signaling cells to produce a specific response only in target cells that have receptors for the signaling molecules

• The signal represents information that is detected by specific receptors and converted to a cellular response, which always involves a chemical process

• This conversion of information into a chemical change is known as signal transduction

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The general pattern of cellular communication

Release

Binding of the signaling moleculeby a receptor protein leading to its

activation

Specific changes in cellular function, metabolism or

development

Synthesis of signaling molecule

Transport

Initiation of cellular signal transduction pathways by the

activated receptor

Removal of the signal and termination of the cellular response

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• The majority of receptors are activated by binding of secreted or membrane-bound molecules

• Some receptors, however, are activated by changes in the concentration of a metabolite (e.g., oxygen or nutrients) or by physical stimuli (e.g., light, touch, heat)

• based on the distance over which the signal acts, signaling by soluble extracellular molecules can be classified into three types: endocrine, paracrine or autocrine

• In endocrine signaling, classic hormones (from the Greek for “set in motion”) travel through the blood and other extracellular fluids to act on target cells far from their site of synthesis

• In paracrine signaling, signaling molecules affect cells in close proximity

neurotransmitters and local hormones

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• Autocrine signaling refers to the situation in which cells respond to substances that they themselves release

some growth factors, PAF, secretions of tumor cells• Some signaling molecules can be short or long range

mediators e.g. epinephrineo Integral membrane proteins can also serve in signaling:

bind with receptors on adjacent cells cleavage of the exoplasmic portion of the proteins

and the fragment acts as a soluble signaling molecule

Features of signal transduction1.Specificity – signaling molecule and receptor are

complementary through non-covalent interactions

even when there are receptors, the subsequent molecules may be absent

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2.Sensitivity –very high affinity between ligand and receptor ligand-receptor interaction may show cooperativity in

some cases the original signal brought by the primary messenger is

amplified by enzyme cascades: the number of activated mediators shows a progressive increase

the constant presence of a signal leads to the desensitization of receptors; sensitivity is restored when the level of the stimulus falls below a certain threshold

there may also be upregulation or downregulation of receptors on the surface of the cell

3.Integration – the ability of the system to receive multiple signals and produce a unified response appropriate to the needs of the cell or organism Signaling pathways interact with each other at different

levels

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• In terms of their precursors , signaling molecules fall into six groups:1. peptides – the largest group; comprising of most classic hormones, growth factors and cytokines

vary from small peptides (TRH = 3 amino acids) to big ones (PTH = 84 a a)

2. steroids –hormones of the adrenal cortex, and the gonads

3. amino acids and their derivatives – thyroid hormones and several neurotransmitters

4. fatty acid derivatives –eicosanoids, PAF5.vitamins– vitamins A and D6. nucleotide derivatives – adenosine, ADP, ATP

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The synthesis of peptide signaling molecules• Like all proteins, they are synthesized by DNA

transcription and mRNA translation• The primary transcript of the hormone may code for an

inactive prohormone which undergoes proteolysis to give the active hormone

e.g. preproinsulin → proinsulin →insulin; the prohormone of ACTH is cleaved to active ACTH, β-endorphin and melanocyte stimulating hormone (MSH)

• Some peptide hormones undergo glycosylation in the Golgi

• The individual components of dimeric peptide hormones (TSH, hCG, LH, FSH) are encoded by separate genes

they share a common α subunit but possess specific β subunits

o Steroid hormones also may need activation; e.g. testosterone has to be reduced to 5α dihydrotestosterone

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Hormone transport• Peptides are hydrophilic and travel in the blood

unbound• Steroid hormones and T4 are complexed with specific

binding proteins like cortisol binding globulin (CBG), sex hormone binding globulin (SHBG),…

• Albumin may serve in non-specific steroid hormone transport

Hormone inactivation • Inactivation of molecules may be specific (enzyme-

based) or non-specific mechanisms • Steroid hormones are biotransformed in the liver and

removed through the urine• The products of MAO and COMT action on

catecholamines and other neurotransmitters are removed through the urine as they are or may be further modified by the liver before excretion

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• Peptide hormones are completely degraded and the amount of active peptide hormones present in the urine is negligible

• Eicosanoids are inactivated through the chemical modification of groups essential for their activity and they ultimately give water-soluble products like dicarboxylic acids

The hierarchy of neuronal and hormonal signals • Most of the classical endocrine glands are functionally

hierarchical; a notable exception being pancreatic islets• A hormone secreted from one gland activates another gland

to produce another hormone, which in turn activates the target tissue –an axis is formed

• Such axes constitute an ‘amplification cascade’ (even before reaching the level of enzymatic cascades) allowing the primary signal to be enhanced several fold

• Negative feedback regulation at different levels; positive feed back rare e.g. increasing production of oxytocin by the pituitary in response to cervical stretch during childbirth

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Categories of signaling mechanisms• There are six basic types of signaling mechanisms:

gated ion channels receptor enzymes

receptors that activate enzymes through G-proteins receptors that activate enzymes without the

involvement of G-proteins intracellular receptors

adhesion receptors that recognize signaling molecules bound with cells

o Please read a bit about ligand-gated and voltage-gated channels; the role of integrins in the extracellular matrix

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RECEPTOR ENZYMES • They usually have a ligand-binding extracellular domain

and a cytosolic domain with enzymatic activity • The two domains are connected by a single

transmembrane domain • The enzyme can have tyrosine kinase, serine/threonine

kinase or guanylyl cyclase activities Tyrosine kinases

The insulin receptor • Is an α2β2 dimer bound by disulfide bridges • The insulin binding site is on the α subunits • The enzymatic activity is in the cytosolic part of the β

subunits• First there is auto(cross)phosphorylation of tyrosine

residues in the C-termini of the subunits• Then comes the phosphorylation of other proteins

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• The main protein phosphorylated by the insulin receptor is called insulin receptor substrate 1 (IRS-1)

• The phosphorylated IRS-1 acts like a ‘docking’ site for a system of adaptor proteins which conduct the signal further

• The signal maybe destined for the cytosol or the nucleus The cytosol • Phosphoinositide 3-kinase (PI-3k) binds IRS-1

the phosphotyrosine residues on IRS-1 are recognized by an SH2 (Src homology 2) domain on PI-3K

• PI-3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-bisphosphate (PIP3)

• PIP3 binds protein kinase B (PKB); the binding favors the phosphorylation and activation of PKB by PDK 1

• PKB phosphorylates Ser and Thr residues on target proteins like glycogen synthase kinase (GSK ) 3, and Glut 4

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o PKB is also involved in the pathway activated by Δ9-tetrahydrocannabinol (THC)

THC binds to the receptors of endocannabinoids (such as anandamide ) which is synthesized from arachidonate and phosphatidylethanolamine

• In addition to the phosphorylation of proteins, insulin exerts many of its effects through dephosphorylation Dephosphorylation activates glycogen synthase

and pyruvate dehydrogenase but it inactivates glycogen phosphorylase and hormone-sensitive lipase

It is unclear how insulin activates phosphoprotein phosphatases

THC

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the PKB signal can be terminated by a phosphatase that removes the phosphate at the 3 position of PIP3

The nucleus • Insulin exerts its effects on genetic expression (of

enzymes that control metabolism and the cell cycle) through a different pathway

• The SH2 domain of a protein known as Grb 2 attaches to phosphotyrosine residues on IRS-1

• Through its SH3 domain Grb 2 attaches to Sos• Sos catalyzes the replacement of GDP for GTP on Ras• 3 kinases – Raf-1, MEK and ERK – are consecutively

activated • ERK enters the nucleus and activates proteins that

serve as transcription factors • ERK is a member of a family of enzymes known as

mitogen activated protein kinases (MAPK)

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• there are many other receptor enzymes which have receptor tyrosine kinase activities

epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF),…

• EGF is a small, soluble peptide hormone that binds to cells in the embryo and in skin and connective tissue in adults, causing them to divide

It is synthesized as an integral plasma membrane protein

Membrane-bound EGF can bind to and signal an adjacent cell by direct contact

Cleavage by an extracellular protease releases a soluble form of EGF, which can signal in either an autocrine or a paracrine manner

• PDGF stimulates growth and tissue repair in an injured region

• Except the insulin receptor, the other receptors in the RTK group dimerize upon the binding of ligand

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Receptor Serine/Threonine kinases • The group includes receptors for members of the

transforming growth factor β (TGF β) superfamily • Isoforms of TGFβ superfamily are inhibitors of proliferation• Loss of TGFβ receptors or certain intracellular signal

transduction proteins in the TGFβ pathway, thereby releasing cells from this growth inhibition, frequently occurs in tumors

• TGFβ also promotes expression of cell-adhesion molecules and extracellular-matrix molecules

• TGFβ signals certain types of cells to synthesize and secrete growth factors that can overcome the normal TGF β -induced growth inhibition; this explains why TGF was originally detected as a growth factor

• The TGFβ receptor complex is composed of two different single membrane-spanning receptor subunits (type I and II)

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• TGFβ binds to a type II receptor and the activated type II receptor recruits a type I receptor which it phosphorylates at a serine residue, forming an activated receptor complex.

• The type I receptor then binds a receptor-specific Smad protein (called R-Smad) which it phosphorylates at serine residues

• The phosphorylated R-Smad undergoes a conformational change and dissociates from the receptor

• R-Smad forms a complex with another member of the Smad family, Smad 4 (Smad 4 is known as the common Smad, Co-Smad, and is not phosphorylated)

• The Smad complex, which may contain several Smads, translocates to the nucleus, where it activates or inhibits the transcription of target genes

• Receptors for different ligands bind different Smads, which bind to different sites on DNA and regulate the transcription of different genes

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• Within the nucleus R-Smads are being continuously dephosphorylated, which results in the dissociation of the R-Smad/co-Smad complex and export of these Smads from the nucleus ----- signal termination

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Guanylyl cyclase activity• Atrial natriuretic factor (ANF), NO (EDRF),

intestinal guanylin • the binding of signaling molecules leads to the synthesis

of cGMP from GTP• cGMP activates cGMP-dependent protein kinase also

known as protein kinase G (PKG) • PKG phosphorylates various proteins at Ser and Thr

residues leading to the physiological effects of the mediators

guanylin increases the secretion of Cl_ in the intestine the receptor for guanylin can also bind the toxin

produced by E. coli and other bacteria• The signaling is terminated when cGMP is converted to

GMP by a variety of phosphodiesterases

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G-PROTEIN-COUPLED RECEPTORS (GPCR)• The family includes receptors for several hormones and

neurotransmitters, light-activated receptors (rhodopsins) in the eyes, odorant and taste receptors

• The defining features are: a plasma membrane receptor with seven membrane-

spanning regions (7tm; serpentine receptor) with the N-terminal segment on the exoplasmic face and the C-terminal segment on the cytosolic face

an enzyme in the plasma membrane that generates an intracellular second messenger

a guanosine nucleotide–binding protein (G protein)

• G-proteins contain three subunits:α, β and γ• During intracellular signaling the β and γ subunits remain

bound together and are usually referred to as the Gβγ

subunit

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• The Gα subunit is a GTPase switch protein that alternates between an active (on) state with bound GTP and an inactive (off) state with bound GDP

• Binding of the receptor by the appropriate signaling molecule activates the G- protein and the activated G-protein in its turn activates an associated effector protein

The effector protein may be inhibited in some cases • The effector protein may be:

adenylyl cyclase (AC) that converts ATP to cAMP phospholipase C (PLC) which converts PIP2 to

diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3)

• cAMP, IP3 and DAG are second messengers; so is cGMP• The second messengers activate a range of proteins leading

to different physiological effects • Hydrolysis of GTP to GDP (by an intrinsic ATPase) causes Gα to

dissociate from the effector and reassociate with the Gβγ

subunit

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• the mechanism of β1 and β2-adrenergic receptors is the prototype for the action of GPCRSignaling through the Gs protein leads to lipolysis

in the adipose, glycogenolysis in the liver and the muscles, contraction of cardiac muscles

In the liver glucagon and epinephrine bind to different receptors, but both receptors interact with and activate the same Gs

• The α1-adrenergic receptor is coupled to a Gi protein that inhibits AC

• The Gq protein coupled to the α2-adrenergic receptor activates a different effector enzyme that generates different second messengers

• cAMP exerts its effects through the activation of cAMP-dependent protein kinase also known as protein kinase A (PKA)

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• Inactive PKA is a tetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits

• Each R subunit has two distinct cAMP-binding sites• binding of cAMP to both sites in an R subunit leads to

release of the associated C subunit unmasking its catalytic site and activating its kinase activity

• PKA phosphorylates and inactivates glycogen synthase • PKA also phosphorylates glycogen phosphorylase

kinase; glycogen phosphorylase kinase phosphorylates glycogen phosphorylase

• The net effect is an increase in the amount of free glucose

• Phosphoprotein phosphatases (PP) reverse the effect of PKA by removing the phosphates added to different enzymes through the action of PKA

• PKA regulates the activity of PP :PKA phosphorylates and activates an inhibitor of PP

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• Epinephrine levels as low as 10-10 M can stimulate liver glycogenolysis

10-6 M cAMP can be produced

The amount of glucose released can reach 10-2 M

• In skeletal muscles the ratios of the concentrations of PKA:GPK:GP is 1:10:240

SIGNAL AMPLIFICATION

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• Just like in the case of cGMP, phosphodiesterase converts cAMP to AMP terminating the signaling

• Insulin activates phosphodiesterase• Methylxanthines such as caffeine and

theophyline inhibit phosphodiesterase; cAMP persists and mobilization of fuels increases

• In the continuous presence of epinephrine, the receptors are desensitized

The receptor is phosphorylated both by β-adrenergic receptor kinase (βARK) and PKA

The phosphorylation creates a binding site for the protein β-arrestin (βarr)

The binding of β-arrestin inhibits the interaction of the receptor with G-proteins; it also facilitates the removal of the receptors from the cell membrane by endocytosis

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• Toxins produced by Vibrio cholerae, which causes cholera, and several other enteric bacteria alter the permeability barrier of the intestinal epithelium by altering the composition or activity of tight junction

• The cholera A toxin catalyzes the transfer of ADP-ribose from NAD+ to the subunit of Gs blocking its GTPase activity; Gs will be permanently activated

• The continuous activation of AC of intestinal epithelial cells and the subsequently high levels of cAMP trigger constant secretion of Cl-, HCO3

- and water into the intestinal lumen • Bordetella pertussis, a bacterium that commonly infects

the respiratory tract, is the cause of whooping cough • Pertussis toxin catalyzes a modification of G i that prevents

release of bound GDP, thus locking Gi in the inactive state• This inactivation of Gi leads to an increase in cAMP in

epithelial cells of the airways, promoting loss of fluids and electrolytes and mucus secretion

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cAMP and genetic expression• Some of the catalytic subunits of PKA translocate to the

nucleus and phosphorylate serine-133 on cAMP-response element binding (CREB) protein

• Phosphorylated CREB associates with the co-activator CBP/P300

• The CREB/ CBP/P300 complex binds to cAMP-response elements (CRE) on the DNA leading to the transcription of genes for proteins involved in various processes

e.g., the production of somatostatin (a peptide that inhibits the release of various hormones); gluconeogenetic enzymes in the liver

• This means that cAMP, in addition to the relatively swift modulation of the activities of various enzymes, also activates a slower response pathway, gene transcription

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OTHER G-PROTEIN-MEDIATED PATHWAYS • Cardiac muscarinic acetylcholine receptors activate

an inhibitory G-protein that opens K+ channels• Binding of acetylcholine causes a hyperpolarization that

lasts for several seconds• The signal from the activated receptor is conducted to the

effector protein (the K+ channel) by the Gβγ subunit rather than the Gα subunit

• Rhodopsin, a G-protein–coupled receptor that is activated by light is found in the rod cells

• Rhodopsin has two covalently bound parts: 11-cis-retinal (the light-absorbing pigment) and the 7tm opsin

• The G-protein found associated with rhodopsin is known as transducin (Gt)

• Upon absorption of a photon, the retinal moiety of rhodopsin is very rapidly converted to the all-trans isomer, causing a conformational change in the opsin portion that activates it

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Pit stopThe Vitamin A group• Also known as retinol, is synthesized from isoprene• Usually occurs in the form of esters – retinyl esters • Retinol can be absorbed in the diet from animal sources

or synthesized from β-carotene from plant sources• Once ingested, preformed vitamin A or β-carotene and

its analogs are released from proteins by the action of proteolytic enzymes in the stomach and small intestine

• The free carotenoids and retinyl esters aggregate in fatty globules that enter the duodenum

• Digestion, absorption and transport to the liver related to that of lipids

• Retinol delivered to the rods is oxidized by a specific retinol dehydrogenase to become all-trans retinal and then converted to 11-cis retinal by retinal isomerase

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• The resulting form in which opsin is covalently bound to all-trans-retinal is called metarhodopsin II or activated opsin

• Metarhodopsin II activates transducin metarhodopsin II is unstable and spontaneously

dissociates, releasing opsin and all trans-retinal, thereby terminating visual signaling

In the dark, free all-trans-retinal is converted back to 11-cis-retinal, which can rebind opsin, reforming rhodopsin

• In the dark, the membrane potential of a rod cell is about -30 mV, considerably less than the resting potential (-60 to -90 mV) typical of neurons and other electrically active cells

• As a consequence of this depolarization, rod cells in the dark are constantly secreting neurotransmitters that inhibit bipolar neurons

• The depolarization of the rod cells is because of non-selective ion channels that admit Na+ and Ca2+ , as well as K+

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• The more photons absorbed by rhodopsin, the more channels are closed, the fewer Na+ ions cross the membrane from the outside, the more negative the membrane potential becomes, and the less neurotransmitter is released

• This change is transmitted to the brain where it is perceived as LIGHT

• A single photon absorbed by a resting rod cell produces a measurable response, a decrease in the membrane potential of about 1 mV; humans are able to detect a flash of as few as five photons

• The key transducing molecule linking activated opsin to the closing of ion channels is cGMP

• Rod outer segments contain a high concentration of cGMP• Light absorption by rhodopsin induces activation of a

cGMP phosphodiesterase• The light-induced decrease in cGMP leads to the closing of

cGMP-gated channels

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• Signal termination is by the rapid hydrolysis of the GTP to GDP

• Rhodopsin is desensitized by rhodopsin kinase which is structurally and functionally similar to βARK

o Three types of cone cells are specialized to detect light from different regions of the spectrum, using three related receptors

o Olfactory and gustatory receptors have a similar mechanism of signal transduction as photoreceptors

G-protein–coupled receptors that activate phospholipase C

• Instead of cAMP, the second messengers in this case are derived from phosphatidylinositol (PI)

• The G-proteins involved are denoted as Gq and Go

• PIP2 is cleaved by the plasma-membrane–associated PLC to generate 1,2-diacylglycerol (DAG), a lipophilic molecule that remains associated with the membrane, and inositol 1,4,5-trisphosphate (IP3) which diffuses into the cytosol

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* Please contrast this with the formation of PIP3

from PIP2 in insulin signaling

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• Most intracellular Ca2+ ions are sequestered in the mitochondria, the lumen of ER, the Golgi, nucleus, vesicles in the cytoplasm known as calciosomes, granules

• IP3 binding induces opening of a Ca2+ channels on the membrane of the ER, allowing the entry of ions into the cytosol

• The rise in the cytosolic Ca2+ level is transient because Ca2+ ATPases located in the plasma membrane and ER membrane actively pump out Ca2+

in addition, IP3 is inactivated by hydrolysis to inositol 1,4-bisphosphate

• Up to a certain point cytosolic Ca2+ ions increase the affinity of the channel receptors for IP3, resulting in a greater release of stored Ca2+ ; higher concentrations of cytosolic Ca2+, however, exert the opposite effect

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• DAG, in association with Ca2+ activates protein kinase C (PKC)

• In the absence of hormone stimulation, PKC is present as an inactive cytosolic protein

• A rise in the cytosolic Ca2+ level causes PKC to bind to the cytosolic leaflet of the plasma membrane, where the membrane-associated DAG can activate it

o DAGs usually contain arachidonate for eicosanoid synthesis• A small cytosolic protein called calmodulin functions as a

multipurpose switch protein that mediates many cellular effects of Ca2+

• Calmodulin may exist as an individual monomeric protein or as a subunit of a multimeric protein

• The binding of Ca2+ to four sites on calmodulin causes a conformational change that permits Ca2+/calmodulin to bind various target proteins, thereby switching their activity on or off

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• Examples of enzymes activated by Ca2+ /calmodulin complex include Ca2+/calmodulin – dependent protein kinases, glycogen phosphorylase kinase, myosin light chain kinase, cAMP phosphodiesterase, nitric oxide synthase

• In hepatic cells, PKC inhibits glycogen synthase (GS)• GS is also inhibited by glycogen phosphorylase kinase (GPK)

and Ca2+/calmodulin-dependent protein kinase (CDPK)The action of GPK is in addition to its activation of

glycogen phosphorylase• This means that epinephrine exerts a dual incremental effect

on hepatic glucose production acting through its α1 and β receptors

• Calcium increases glycogenolysis in the muscles too• But the effects of Ca2+ in muscle result principally from the

release of Ca2+ from the sarcoplasmic reticulum after neural stimulation, and not from epinephrine-stimulated release

o Moreover, muscle glycogen phosphorylase is activated by AMP

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Glycogenolysis in the

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Glycogenolysis in the muscles

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• In certain cells, the rise in cytosolic Ca2+ following the release of IP3 leads to activation of specific transcription factors

• In some cases, PKC and Ca2+ /calmodulin-dependent protein kinases phosphorylate transcription factors; In other cases, Ca2+/calmodulin activates a phosphatase that removes phosphate groups from transcription factors

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RECEPTORS THAT ACTIVATE ENZYMES WITHOUT THE NEED FOR G-PROTEINS

• receptors that have no intrinsic protein kinase activity but, when occupied by their ligand, bind a soluble tyrosine kinase

• the receptors for cytokines fall under this category• the soluble tyrosine kinases have a family name of Janus

Kinase (Jak)• Similarities with receptor tyrosine kinases:

Receptors form dimers upon ligand binding (except for the insulin receptor, which is a dimer from the outset)

Binding of signaling molecules causes one of the poorly-active kinases to phosphorylate a tyrosine residue at the activation lip of the second kinase

Several other tyrosine residues of the receptor will then be phosphorylated; these phosphotyrosines (and for insulin, IRS-1) will be used as docking sites for various signal-transduction proteins

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• The receptor phopshotyrosines resulting from the action of Jak are recognized by the SH2 domain of STAT (signal transducer – activators of transcription)

• Jak phosphorylates the STAT bound with the receptor• A phosphorylated STAT dissociates from the receptor,

and two phosphorylated STAT proteins form a dimer in which the SH2 domain on each binds to the phosphotyrosine in the other

• STAT dimers move into the nucleus, where they bind to specific DNA sequences activating transcription

• In erythroid progenitors stimulation by erythropoietin (EPO) leads to activation of STAT5

• The protein whose synthesis is induced by active STAT5 prevents apoptosis of these progenitors allowing them to proliferate and differentiate into erythroid cells

• Activated JAK can also trigger, through Grb2, the MAPK cascade

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• Short term termination of Jak/STAT signaling is effected through the removal of the phosphate from tyrosine of the activation lip of Jak

•SOCS proteins terminate signaling over longer time periods

Binding of SOCS to phosphotyrosine residues on receptors or Jak blocks the binding of other signaling proteins

SOCS can also target Jak for degradation by proteasomes

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A few points on cytokines…• Cytokines form a family of relatively small, secreted

proteins (~160 amino acids) that control many aspects of growth and differentiation of specific types of cells

• During pregnancy prolactin induces the differentiation of the immature epithelial cells lining the mammary gland to differentiate into mature milk-secreting cells

• Interleukin 2 (IL-2) and IL-4 are essential for proliferation and functioning of the T and B cells of the immune system

• Interferon-α produced and secreted by many types of cells following viral infections

• Granulocyte colony stimulating factor (G-CSF), thrombopoietin and erythropoietin induce the differentiation progenitor cells into various types of blood cells

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SIGNALING THROUGH INTRACELLULAR RECEPTORS• Lipid-soluble hormones like steroid hormones, thyroid

hormones, retinoic acid and vitamin D can diffuse through plasma and nuclear membranes and interact directly with the transcription factors that they control

• The intracellular receptors for most of these lipid-soluble hormones, which constitute the nuclear-receptor superfamily, function as transcription activators when bound by ligands

• The sites on the DNA that bind nuclear receptors are called hormone response elements (HRE)

• Hours or days may be needed for the effects of the hormones to be felt; some exceptions

• There are two types of intracellular receptors:1. Nuclear receptors –for thyroid hormones, retinoic

acid , vitamin D, … • in the absence of ligand they are found as dimers

bound to HREs repressing transcription

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• Sources of ligands for nuclear receptors: from the classical endocrine pathway (thyroid hormones) intracellular synthesis from inactive precursors

(retinoic acid from retinol) fully synthesized inside the same cell in which

receptor activation takes place (prostaglandin J2 )2. Cytosolic receptors –binding of ligand directs their

translocation to the nucleus; for steroid hormones• in the absence of hormone cytosolic receptors are found

as dimers in the cytosol as large protein aggregates complexed with inhibitor proteins such as the HSP family

• When the hormone binds, the receptor undergoes a conformational change and dissociates from the proteins, exposing a nuclear translocation signal

• the complex translocates to the nucleus, where it binds HREs

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o Steroid hormone receptors can be useful targets for drugs• The synthetic progesterone agonist norethindrone is

widely used as a component of contraceptive pills• The drug tamoxifen is used to treat breast cancer in

some types of the cancer where the division of the cancerous cells is dependent on the continuous presence of estrogen

• Mifepristone also known as RU 486 is a powerful antagonist of both progesterone and glucocorticoids; it is an effective emergency contraceptive agent

• Cortisone and synthetic analogs such as prednisolone and dexamethasone are widely used anti-inflammatory agents

Either the alcohol or the aldehyde form of vitamin A has an essential function in reproduction, whereas bone growth and maintenance of mucous secretions requires only retinoic acid

Retinol and retinoic acid, reduce the incidence of experimentally-induced cancer

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CELL BIRTH, CELL DEATH AND CANCER• Proliferating cells undergo a cycle of division –the cell

cycle –which lasts approx. 24 hours in mammalian cells in cell culture

• Fully differentiated animal cells divide rarely; they are said to be quiescent or in the G0 phase, in which they can remain permanently

• The cell cycle is divided into four stages: S (synthesis) phase –the DNA is replicated to

produce copies for both daughter cellsG2 phase (G refers to ‘gap’) –new proteins are

synthesized and the cell approximately doubles in size M (mitotic ) phase –the maternal nuclear envelope

breaks down, matching chromosomes are pulled to opposite poles of the cell, new nuclear envelopes are formed, and cytokinesisG1 – a waiting period before entering into another cycle

of division

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• In embryonic or rapidly proliferating tissue, each daughter cell divides again after G1

• In other types of tissues, cells enter G0 which may last hours, days or the lifetime of the cell

• Some G0 cells return to the G1 phase again under the influence of mitogens

• Together, the G1, G0, S and G2 phases are referred to as the interphase, which alternates with the short M phase

• The various cell cycle events are highly coordinated to occur in a defined order and with an exact timing, requiring precise control mechanisms

• Intrinsic control mechanisms are of two types:1. Constitutive –operational in every cell cycle ensures the

ordering of individual steps 2. Induced –active only when defects are detected in cell cycle

events • The points where control mechanisms function are known as

checkpoints

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• In addition to the built-in control mechanisms, the cell is also subject to a number of external controls in the form of nutrients and growth factors

• Two processes are central to the external control of cell cycle regulation:

oscillating changes in the activity of the cell cycle machinery, with protein kinases as the most important component

specific proteolysis of cell cycle regulators• The proteins involved are cyclin-dependent

protein kinases (CDKs), cyclins, other kinases and phosphatases and inhibitors of cyclin-dependent protein kinases (CKIs)

• The CDKs are Ser/Thr kinases that must associate with the corresponding cyclin in order to be active

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• In each phase of the cell cycle specific CDKs associate with equally phase-specific cyclins

• The activated complex in turn phosphorylates transcription factors, which finally lead to the formation of the proteins that are required in the cell cycle phase concerned (enzymes, cytoskeleton components, other CDKs and cyclins)

• The activity of the CDK–cyclin complex is then terminated again by the proteolytic degradation of cyclins

• The G1–S transition is particularly important for initiating the cell cycle

• The daughter cells produced by cell division must reach a critical size in the course of G1 phase before S phase can commence ---- internal control

• Mitogens initiate progression through G1 by stimulating the synthesis of G1 cyclin-CDK complexes (start cyclin-CDK complexes; cyclin D-CDK4/6) ---- external control

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• The activity of these and other cyclin-CDK complexes is regulated by phosphorylation and dephosphorylation and through the binding of inhibitor proteins

• Once mitogens have acted for a sufficient period, the cell cycle continues through mitosis even when they are removed

• The point in late G1 where passage through the cell cycle becomes independent of mitogens is called the restriction Point (R)

• The G1 cyclin-CDK complex by phosphorylating the protein pRb (Rb=retinoblastoma) releases the transcription factor E2F previously bound to pRb

• This activates the transcription of genes needed for DNA replication and cyclins and CDKs for later stages of the cycle

• If the DNA is damaged the protein p53 initially delays entry into the S phase

• If the DNA repair is not successful, p53 forces the cell into apoptosis

88The workings of the start cyclin-CDK complex

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• S-phase cyclin-CDK complexes (cyclin A/E –CDK2) phosphorylate regulatory sites in the proteins that form DNA pre-replication complexes

• Phosphorylation of these proteins not only activates initiation of DNA replication but also prevents reassembly of new pre-replication complexes

• Because of this inhibition, each chromosome is replicated just once during passage through the cell cycle

• Mitotic cyclin-CDK complexes (cyclin B-CDK1) are synthesized during the S phase and G2, but their activities are held in check by phosphorylation at inhibitory sites until DNA synthesis is completed

• Activated mitotic cyclin-CDK complexes phosphorylate multiple proteins that promote the chromosome condensation, retraction of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate

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• Catastrophic genetic damage can occur if cells progress to the next phase of the cell cycle before the previous phase is properly completed

• There are specific checkpoints for the correct replication of all chromosomes, the proper segregation of daughter chromosomes, the correct assembly of the mitotic spindle, the ‘health’ of the DNA

• Cells with damaged DNA are arrested before they enter the M-phase with the help of tumor suppressor proteins such as pRb and p53

• p53 evolved to induce apoptosis when there is extensive DNA damage, presumably to prevent the accumulation of multiple mutations that might convert a normal cell into a cancer cell

• Nearly all cancer cells have mutations in both alleles of the p53 gene or in the pathways that stabilize p53 in response to DNA damage

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How is apoptosis carried out?• Growth factor deprivation (detachment), DNA

damage (p53) and “death” signals from outside are the main causes of apoptosis

• The effector proteins in the apoptotic pathway are enzymes called caspases; caspases are synthesized as procaspases

• The release of cytochrome C from the mitochondria is crucial for the apoptotic process Cytochrome C helps to activate procaspase 9 and

caspase 9 in turn activates procaspase 3 Caspase 3 cleaves other downstream procaspases

and apoptotic substrates• Apoptosis can be activated by external ligands that bind

to and activate receptor systems known as death receptors

• The death receptors are transmembrane receptors that belong to the superfamily of tumor necrosis factor (TNF) receptors

93Death in the absence

of growth factors

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• Members of the family are characterized by a ligand binding extracellular domain and an intracellular domain known as the death domain (DD)

• The ligands for the receptors include: Tumor necrosis factor (TNF) –released by macrophages,

triggers the cell death and tissue destruction seen in certain chronic inflammatory diseases

The Fas ligand –is a cell-surface protein expressed by activated natural killer cells and cytotoxic T lymphocytes; this signal can trigger death of virus-infected cells, some tumor cells, and foreign graft cells

• The binding of TNF to its receptor leads to the release of an inhibitory protein (silencer of death domain, SODD)

• Subsequently, the adaptor protein TRADD (TNF-associated death domain) associates with the intracellular domain of the receptor and recruits additional adaptor proteins including FADD (Fas-assoc...) and inhibitors of NFκB kinases

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FADD activates procaspase 8 (initiator caspase) …apoptosis of non-immune cells

Activation of NFκB leads to the proliferation of immune cells

• FAS ligand, on the other hand, recruits FADD only

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Oncogenes• Onkos, “mass” (mass generators) • Mutations in two broad classes of genes have been

implicated in the onset of cancer: proto-oncogenes and tumor suppressor genes

• Proto-oncogenes are activated to become oncogenes by mutations that cause the gene to be excessively active in growth promotion

• Tumor suppressor genes normally restrain growth, so damage to them allows inappropriate growth

• Cancer commonly results from mutations that arise during a exposure to carcinogens, which include certain chemicals and ultraviolet radiation

• Cancer-causing mutations occur mostly in somatic cells, not in the germ-line cells

• Certain inherited mutations, which are carried in the germ line, increase the probability of cancer occurring

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• Cellular genetic material that has been incorporated into the genome of viruses may undergo mutation to give v-onc

• Oncogenes normally present in the cells are designated as c-onc

• Proto-oncogenes control normal cell growth and division• These genes encode proteins that are growth factors, growth

factor receptors, signal transduction proteins, transcription factors, cell cycle regulators and regulators of apoptosis

• The mutations in oncogenes are usually gain-of-function mutations; either a more active protein is produced or an increased amount of the normal protein is synthesized

• Tumor suppressor genes protect against uncontrolled cell proliferation

• A transforming mutation in these protective genes results in a loss of activity or a decreased amount of the gene product

• A normal cell that has undergone changes to become a cancerous cell is said to have been transformed

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• The hallmarks of transformation are: Clones of a single cell Loss of contact inhibition Growth factor- independence Immortality Increased angiogenesis Metastasis

• A single activated oncogene does not usually lead to a loss of growth control

• The transformation process has three stages: initiation, promotion and progression

Transformation is initiated through genetic change in proto oncogenes while promoters act without changing the structure of DNA

• Over the course of time mutations and regulation defects may accumulate in a single cell –progression

• If the immune system does not succeed in eliminating the transformed cell, it can over the course of months or years grow into a macroscopically visible tumor

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• A feature common to all oncogenes is the fact that they code for proteins involved in signal transduction processes

• The genes are designated using three-letter abbreviations that usually indicate the origin of the viral gene and are printed in italics (e.g., ras); the names of the protein products are not italicized but are capitalized (Ras)

Groups of oncogenes –genes for:1.Ligands such as growth factors and cytokines 2.Membrane receptors for growth factors and hormones 3.GTP-binding proteins like Ras 4. Nuclear receptors which serve as transcription factors

for proteins such as Erb5.DNA binding proteins that are transcription factors for

enzymes involved in cell proliferation; Myc, Fos, Jun, … 6.Protein kinases such as Raf which is activated by Ras * Nuclear tumor suppressors such as pRb and p53 are

referred to as anti-oncogens

signal transduction

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signal acts

specific receptors

signal transductioneven

signal leads

original signal

moleculesthe signal

downregulation of receptors

majority of receptors