Mechanism of hormone and

neurotransmitter action

Types of membrane and andintracelular receptors

Department of Biochemistry FM MU 2011 (E.T.)

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– Regulation of metabolic events within particular compartment(cellular organelle) that depends only on interactions betweenmolecules in the compartment;

– regulations that occur within complete cells without any regard toextracellular signals, in which proteosynthesis and transport acrossmembranes that separate individual compartments have the importantroles have;

– regulations that are consequences of communication between cellsin particular tissues, organs, or the whole organism, depending onextracellular signals – neurotransmitters, hormones, cytokines, andother signal molecules.

Control of metabolism

Numerous metabolic pathways are controlled usually in only one orfew check-points (rate-limiting steps) by more than one differentmechanisms.

These formal levels of metabolism control mostly overlap.

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Mechanism of hormone andneurotransmitter action

Signal molecule types in neurohumoral regulations:

Signal molecule Origine

HORMONES secreted by endocrine glands, by dispersed

glandular cells (eicosanoids by many othercellular types)

NEUROHORMONES secreted by neurons into the blood circulation

NEUROTRANSMITERS secreted by neurons at nerve endings

CYTOKINES, GROWTHFACTORS, IKOSANOIDS

secreted by various types of cells

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Action of signal molecules

Action Character of action

endocrine Signal is carried by the blood, may act in the

whole body. Typically hormones

paracrine Signals act within short distances of the site oftheir production

autocrine Signal act on the cells that produce them

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Signal transduction

How cells receive, process, and respond to information from theenvironment?

Reaction of signal molecule with receptor

Membrane receptors

Proteins and small polarsignal molecules (aminoacids, peptides, biogenicamines, eicosanoids)

Intracelular receptors

Nonpolar signal molecules(steroids, iodothyronines,retinoates)

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Interaction of the complex hormone-receptorwith the HRE of nuclear DNA

Nonpolar signal moleculebound to the plasma transport protein

Intracellular receptor

Biological response(the effect is slow, either early or late)

Amplification

Polar signal molecule

Biological response(prompt effect)

Signal transduction

Membrane receptor

Membrane and intracellular receptors

Transport of signal molecule

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Main types of membrane receptors

Receptors – ion-channels (ROC, ligand gated ionophores) serveexclusively as receptors for neurotransmitters (see lecture 7).

Receptors activating G-proteins (heterotrimeric G-proteins), the resultof specific ligand binding is mostly

- stimulation or inhibition of adenylate cyclase,- stimulation of phospholipase C,- stimulation of phosphodiesterase.

Receptors exhibiting intrinsic catalytic activity- guanylate cyclase activity – receptors for natriuretic peptides,- tyrosine kinase activity

- insulin receptor, receptors for insulin-li growth factors (IGF1,2),- dimerizing receptor for epidermal growth factor (EGF).

Receptors cooperating with non-receptor tyrosine kinases(e.g., Janus kinase JAK) – receptors for somatotropin (growthhormone), prolactin, erythropoietin, interferons, interleukins and othercytokines.

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Common structural features :

All of them are seven -helical segmentsthat span the membrane and are connectedby intra- and extracellular hydrophilic andmore divergent loops.

H2N

-COOH

Family of heterotrimeric G-protein-coupled receptors

Binding site for theagonist (alsoaccessory bindingsites for antagonists)

Intracellular domains -binding site for the specificG-protein type.

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Heterotrimeric G-proteins

Proteins binding GDP or GTP

mostly freely membrane-bound (they can move along the innersurface of the plasma membrane).

Subunits , a .

More than 20 different subunits havebeen identified.

Subunits G andG arehydrophobic andnon specific

G subunit is the largest,hydrophilic, it binds GTPor GDP, andIt is specific for particularmechanism of secondmessenger production.

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Trimer G-GDP,G,G

Complexreceptor-specific ligand

Complex receptor-ligand--trimer G-GDP,G,G

Dimer G,G

GTP

GDP

Activatedsubunit G-GTP

PRODUCTION OF THESECOND MESSENGER

Interactionwith the target protein

Pi

Inactivesubunit G-GDP

Dimer G,G

The cycle of G-proteins activation

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Selected types of G protein -subunits

G subunit type Examples of

activating receptors

Effect of activated G

on the target protein

Gs (stimulatory) glucagon

parathyrin

-adrenergic

stimulation of

adenylate cyclase

Gi (inhibitory) somatostatin

2-adrenergic

inhibition of

adenylate cyclase

Gq (activating the PIcascade)

vazopressin V1

endotelin ETA,B

acetylcholine M1

1-adrenergic

stimulation of

phospholipase C

Gt (inhibitory)

(for transducin)

rhodopsine stimulation of

cGMP phosphodiesterase

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Hormone receptors that activate Gs or Gi proteinsstimulates or inhibit adenylate cyclase

Adenylate cyclase, a membrane-bound enzyme, catalyzes the reactionATP cAMP + PPi ; the second messenger is cyclic AMP.

AMP-cyclasereceptor receptorGS Gi

ligand ligand

ATP

cAMP

proteinkinase Ainactive (R2C2)

active proteinkinase A2 C + 2 R(cAMP)2

AMP H2O

*phosphodiesterase

phosphorylations

*Inhibition by caffein, theofyllin

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cAMP action in the cells

C

C R

R

ProteinkinaseA (inactive)

R

R

C

C

Protein

Protein

Protein-P

Protein-P

ADP

ADP

ATP

ATP

ProteinkinaseA (active)

cAMP

Phosphorylation of proteins.

In cytoplasma - mostly metabolic enzymes (rapid response)

In the nucleus – phosphorylation of gene specific transcription factorCREB (cAMP response element-binding protein) (slower response)

AKAPAKAP

They targetactivity tophysiologicalsubstrates

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Localization of cAMP action in specific site ofthe cell

Proteins AKAPs (A kinase anchoring proteins)

Proteins binding proteinkinase A, they target its activity to physiologicalsubstrates (they serve as a scaffold which localize PAK near tosubstrate).

Similar proteins affects also the specific action of phosphatases,phosphodiesterases.

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Phospholipase C

Both reaction products are the secondmessengers:Inositol 1,4,5-trisphosphate opens the Ca2+

channel in ER membrane,diacylglycerol activates proteinkinase C.

Receptors that activate Gq protein stimulatephospholipase C and start the phosphatidylinositol cascade

diacylglycerol

inositol 1,4,5-trisphosphate

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Phosphatidylinositol cascade

phospholipase Creceptor

Gq

specific ligand

PIP2 DG

activation ofproteinkinase C

phosphorylations

increase of [Ca2+]in cytoplasm

Endoplasmic reticulum

Ca2+

IP3 receptors in the membranes of ERact as ligand gated channels for Ca2+ ions

IP3 active proteinkinase C

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Control of metabolism by changes ofcytoplasmic concentration of Ca2+

•Basal concentration of Ca2+ in cytoplasma 1.10-7 mol/l

• Increase to concentration to 1.10-6 rapidly and maximallyagtivates the various Ca2+-regulated cell function

• Increse of Ca2+ can be triggered

by influx of Ca2+ across the plasmaticmembrane (see e.g.smooth muscle contraction)

or by release from intracelular stores (ER,mitochondrias) e.g. IP3 gated Ca 2+ channel inER, or ryanodine receptors in ER/SR of cardiac orskeletal muscle

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Regulatory protein calmoduline

Increased cytosolic Ca2+ activates various calcium-bindingregultory proteins (family of small, Ca 2+ dependent protein).

The most important is calmoduline. It is ubiquitouslyexpressed protein in nearly all cells.

Upon binding of Ca2+ (4 binding sites)calmoduline undergoes conformationalchanges that facilitates its interactionwith downstream signaling proteins,e.g. kinases, phosphatases ect.

Some Ca-calmodulin-dependentkinases are very specific, the other havea broad substrate specifity.

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Receptors with guanylate cyclase activity

After binding of ligand they convert GTP to cGMP

cGMP is the second messenger

It activates proteinkinase G

Two types of receptors:

•membrane-associated

• soluble (cytoplasmic)

NN

NNH

O

NH2

O

O OH

O

OH

PO

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Membrane receptors with guanylate cyclaseactivity

ANP

GTP cGMP + PPi

Protein kinase Ginactive

active protein kinase G (PKG)

Phosphorylation of proteins

Receptors for ANP (atrialnatriuretic factor)

Mainly smooth muscle ofvessels and in kidneys

ANP is producedby cardac atrialtissue in responseto increase ofblood volume orpressure

GMP

phosphodiesterase

H2O

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Soluble receptors with guanylate cyclase activity

NH2 NH2

hem

NO

GTP cGMP

Receptor je dimeric complex andbinds hem

Binding NO to the hem increasescatalytic acitivity guanylate cyclase

NO is generated by the action ofnitroxid synthase (NOS)

NO readily permeates membranes, itcan be produced by one type of the celland rapidly diffuse into neighboring celltypes

Activation ofprotein kinase G

phosphodiesterase

GMP

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Proteinkinase G

cGMP sensitive proteinkinase G

Widely expressed in many cells

It phosphorylates various proteins (enzymes, transportionproteins ect.)

Effect of PKG in smooth muscle

Phosphorylation of proteins:

• inactivation of proteins attenuating Ca2+ release from ER Ca2+

• activation of MLC phosphatase repression of actin-myosin interaction

• decrease of K+-channnels activity decrease of hyperpolarization increased influx of Ca2+ into the cell

Relaxation of smooth muscle

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NO/cGMP signaling in smooth muscle ofblood vessels

cGMP is the key second messenger for induction of vascularsmooth muscle vessels relaxation

vasodilatation and increased bloof flow

NO is produced in endothelial cells by the action of nitroxidsynthase from arginin (activation e.g. by acetylcholine) anddiffuses into adjacent smooth muscle cells

L-Arg ·NO + L-citrullinNO-synthase

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R-O-NO2 nitrit ·NO

Nitroglycerin and other drugs of organic nitratetype are donors of exogenous NO

Glycerol trinitrate

Isosorbide nitrate

Therapy of angina pectoris

Activation of solubleguanylate cyclase

Vasodilatatory effect releases coronary spasmus andnormalizes blood perfusion.

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Inhibition of phosphodiesterase potentiatesthe effect of NO

cGMP GMP

phosphodiesterase

H2O

The drug sildenafil (Viagra) acts as a selective inhibitor of phosphodiesterase 5(PDE5), that is higly expressed in vascular smooth muscle.

Viagra is 80-4000 fold less potent as an inhibitor of other PDE isoforms(including PDE3 that is expessed in heart).

During sexual stimulation NO in corpora cavernosa is released

The level of cGMP is increased

sildenafil prevents decomposition of cGMP

blood vessel in erectile tissue are dilated and blood flow is increased

Several types ofphosphodiesteraseare known,depending of thetype of cell.

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Receptors with tyrosin kinase activity

Common features

• when the signal molecule binds to the receptor, it triggers conformationalchange of the receptor

• this leads to activation of tyrosin kinase activity of the receptor

• the first protein substrate is the receptor itself (autophosphorylation of tyrosinemolecules in the receptor), eventually other proteins are phosphorylated

• phosphorylated tyrosines and other substrates then acts as a recognition oranchoring site for other proteins, adaptor molecules

• adaptor proteins bind to phosphotyrosine residues by SH2 domaines (Srchomology 2 domain) and are also phosphorylated.

• adaptor proteins reacts with other molecules and signal is transmitted throughcascade of other reactions mainly phosphorylation/dephosphorylation, exchangeof quanine nucleotides, changes of conformation etc.

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-S-S--S-S-

-S-S-

Dimeric structure

Binding site for insulin on - subunits

Tyrosin kinase activity on -subunits

Insuline receptor

Binding of insuline to the receptor tyrosin kinase activityautophosphorylation of -subunits and phosphorylation of IRS 1-4 proteins

(insulin receptor substrates 1-4)

-S-S--S-S-

-S-S-

Insulin

-P-P P-P-

IRS1-4IRS1-4 -P

activation and docking ofPI-3-kinase on membraneactivation of phosphoprotein phosphatase-1activation of small G-protein Ras

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Some signaling pathways of insuline

http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7

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Insuline receptor substrates 1-4 are adaptor proteins.If phosphorylated by the insuline-receptor complex, they bind to other proteins

that are activated in this way.

Glykogen synthesis

Phosphorylation of IRS activatesregulatory subunit PIP2-3 kinase

Catalytic subunit of PIP2-3 kinasephosphorylates PIP2 to PIP3

PIP3 activates proteinkinase B (AKT),activation is enabled by PDK(phosphoinositide dependent kinase)

activated AKT difuses to cytoplasmaand phophorylates (inactivates)glykogen synthase kinase

Synthesis of glycogen is activovated(active form is dephosphorylated formof glycogen synthase)

Translocation of glucoseých transporters

Insulin receptor phosphorylates CbI

Complex CbI-CAP translocates to thelipid raft in the membrane

CbI reacts with adaptor adaptor proteinCrk

Crk is associated with C3G

C3G activates TC10 (G-protein)

TC10 activates translocation oftransporters into the plasmaticmembrane

Examples of insulin receptor signaling pathways

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Dimerizing receptor for EGF (epidermal growth factor)containing an intrinsic tyrosine kinase activity

Dimerization of the receptor after binding of ligand

R RR R

-P-PP-

P- SoS

Ras–GTP

Raf

phosphorylationcascade MAP

phosphorylation

Dimerization activates tyrosin kinase activity in cytoplazmatic domain.

Autophosphorylation of the receptor

Adaptor proteins Grb2 (SH-2 domains) bind to phosphorylated sites.

G-protein Ras is activated by the action of SOS protein activation ofMAP-kinase cascade (Ras/MAP-cascade)

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Ras

SoS

Activation of Ras is key step in transmision of signal.Inactive Ras-GDP is converted to Ras –GTP, that activates thenext molecule of the pathway.Inactivation of Ras: hydrolysis of GTP iniciated by otherregulating proteins

Monomeric G-protein that binds GTP and at the same time has GTPaseactivity (structural analog of subunit intrimeric G-protein).

It is activated by binding GTP instead GDP

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Superfamily of Ras proteins

•5 families: Ras, Rho, Arf, Rab, Ran

•They are anchored to lipid membrane by lipid anchors

(myristoyl, farnesyl)

•Monomeric G-proteins that play important role in

regulation of growing, morphogenesis, cell motility,

cytokinesis etc.

•Mutations in Ras genes induce patologic proliferation and

antiapoptosis. About 30 % ofall human tumors involve cells

expressing mutated Ras oncogenes*.

*(Ras genes are named protooncogenes)

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MAP-kinase signaling pathway

(MAP kinase =Mitogen activated protein kinase)

Map-kinase cascade

Ras–GTP

MAP-kinase-kinase-kinase

MAP-kinase-kinase

MAP-kinase

phosphorylation ofcytosolic ormembrane proteins

phosphorylation ofregulatory proteins innucleus, stimulation ofproliferation

ATP

ATP

ATP

ADP

P

ADP

ADP

ADP

ATP

P

P

MAPKKK, Raf

It regulates mainly cell growing anddiferentiation.

MEK

ERK

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Abbr. name Function

PDGF Platelets derived growthfactor

Mitogen for cell of connectivetissue and non differentiatedneuroglia

EGF Epidermal growth factor Mitogen for many cells ofektodermal and mesodermalorigine

FGF-2 Fibroblast growth factor 2 Mitogen for many cells likefibroblasts, endotelhelial cells,myoblasts;

IL-2 Interleukine 2 Mitogen for T-lymphocytes

Mitogens – grow factors stimulating proliferation

Examples of mitogens:

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Receptors activating non-receptor tyrosine kinases

ligand

dimerization

–PSTATSTATSTAT

JAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription)

•Receptor does not have kinase activity, but is associated with tyrosinkinase JAK.

•After binding of ligand receptors dimerize (homodimers or heterodimers)

•Activated JAKs phosphorylates tyrosine residue of the receptor.

•The STAT proteins (signal transducers and activators of transcription) associate

with the receptor and are phosphorylated by JAK.

•STAT phosphates dimerize, translocate to the nucleus, bind to specific DNA

elements and regulate transcription.

–P STAT

–PSTAT

Receptors of cytokines– e.g.interferons,interleukines

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Intracellular receptors of steroid hormones

(and calcitriols), iodothyronines, and retinoates

Receptors are present in cytoplazma or nucleus

Hormon-receptor complexes binds to the specific sites in

DNA and activate the transcription of specific genes

Complex hormon-receptor binds to DNA at HRE (hormon

response element)

Superfamily of steroidal and thyroidal receptors – a family of

structuraly related proteins

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Example of cortisol

hydrophobic molecule penetrates membrane

inactive receptorfor glucocorticoids (GR)forms in the cytoplasmcomplex with hsp 90 dimerand other proteins

active complex receptor-ligand (monomer),hsp 90 and other proteins are released

active complexes form dimers andare translocated into the nucleusthrough nuclear pores

GR

cortisol

CBG

cortisol is transported attached on CBG (corticosteroid-binding protein) in ECF

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Glucocorticoid receptor (GR) – function

Active complex cortisol-receptor binds onto DNA at the specific sequenceGRE (glucocorticoid response element, quite generally HRE – hormoneresponse element)

DNA binding domain

GR

DNA

GRE

cortisol binding domain(hydrophobic pocket)

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GR dimer – intracellular glucocorticoid receptor (dimer)

GRE – glucocorticoid response element

GREB protein – GRE binding protein (a specific transcription factor)

TF IID Pol IICTD

> 1 000 bpmediator proteins

enhancercoactivatorGREB protein

GRE

cortisol-GR dimer complex

promoter

basaltranscriptionapparatus

Active complex cortisol-receptor binds onto DNA at the specific sequenceGRE (glucocorticoid response element, one of the HRE – hormone responseelements).The coactivator and specific hormone response element-binding proteins(GREB-proteins) are also attached. This complex acquires the ability to act asenhancer that supports initiation of transcription on the promoter by means ofmediator proteins.

Initiation of transcription by cortisol