Mechanism of hormone and neurotransmitter action Types of membrane and and intracelular receptors Department of Biochemistry FM MU 2011 (E.T.)
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