Control of metabolism
Mechanism of hormone and neurotransmitter actionMechanism of hormone and neurotransmitter action
Biochemistry II
Lecture 6 2008 (J.S.)
There are three formal levels, in which the control of metabolism is
– Regulation of metabolic events within particular compartment
achieved:
– Regulation of metabolic events within particular compartment
(cellular organelle) that depends only on interactions between
molecules in the compartment;
– regulations that occur within complete cells without any regard to– regulations that occur within complete cells without any regard to
extracellular signals, in which proteosynthesis and transport across
membranes that separate individual compartments have the importantmembranes that separate individual compartments have the important
roles have;
– regulations that are consequences of communication between cells
in particular tissues, organs, or the whole organism, depending on
extracellular signals – neurotransmitters, hormones, cytokines, and
other signal molecules.other signal molecules.
Numerous metabolic pathways are controlled usually in only one or
few check-points (rate-limiting steps) by more than one differentfew check-points (rate-limiting steps) by more than one different
mechanisms.
These formal levels of metabolism control mostly overlap.
2
These formal levels of metabolism control mostly overlap.
Some factors important in control of metabolism:
– Primarily, the equipment of cells with enzymes and other
proteins (the proteome), which is determinated by theproteins (the proteome), which is determinated by the
expression of genes in the given cell type within the given time
period.
– Specific receptors, which enable recognition of extracellular
signal molecules as well as reactions of the cell or body to
changes in the environment.changes in the environment.
– The existence of multiple enzyme forms (isoenzymes) allows to
control particular reaction types by different mechanisms incontrol particular reaction types by different mechanisms in
various compartments, various tissues, or in various time periods.
–- Accessibility of nutrients and other essential substances, on–- Accessibility of nutrients and other essential substances, on
which the energetic state of the cell depends.
3
Three major mechanisms that provide
control of metabolism
1 Regulation of the amount of enzymes (number of enzyme
control of metabolism
1 Regulation of the amount of enzymes (number of enzyme
molecules) present in the cell.
2 Regulation of enzyme activity or activity of regulatory
proteins, on which the activities of enzymes depend.proteins, on which the activities of enzymes depend.
3 Regulation of transport across membranes that separate3 Regulation of transport across membranes that separate
intracellular and extracellular spaces as well as individual
cellular compartments.cellular compartments.
4
– Regulation of proteosynthesis:
1 Regulation of the amount of enzymes
– Regulation of proteosynthesis:
The expression of some genes occurs at a nearly constant rate
(synthesis of constitutive enzymes).(synthesis of constitutive enzymes).
Numerous genes are expressed in response to specific
regulatory signals, expression of some other may be silenced.regulatory signals, expression of some other may be silenced.
The enzymes controlled in this way are adaptable enzymes
(mostly inducible, see chapter Regulation of gene expression).
Regulation of proteosynthesis may occur at the level of gene
amplification, transcription, posttranscriptional hnRNAamplification, transcription, posttranscriptional hnRNA
processing (alternate mRNA splicing), export of mRNA from
nucleus, degradation of mRNA, translation, and posttranslational
modification.modification.
In eukaryotes, expression of genes can be induced by binding of signal
molecules on specific membrane receptors (e.g. growth factors,molecules on specific membrane receptors (e.g. growth factors,
cytokines, and insulin), or by interactions of hydrophobic signal
molecules (steroid hormones, iodothyronines, retinoates) with specific
5
molecules (steroid hormones, iodothyronines, retinoates) with specific
intracellular receptors.
– Regulation of enzyme degradation:
1 Regulation of the amount of enzymes
– Regulation of enzyme degradation:
Rates of degradation of specific enzymes are selectively
regulated, namely of those that catalyze the rate-limiting steps inregulated, namely of those that catalyze the rate-limiting steps in
biochemical pathways or represent important metabolic control
points. Those enzymes are mostly short-lived proteinspoints. Those enzymes are mostly short-lived proteins
(biological half-lives from several minutes to few hours) and their
degradation is provided by cytosolic ubiquitin system, or by otherdegradation is provided by cytosolic ubiquitin system, or by other
systems not yet known.
The susceptibility of an enzyme to proteolytic degradation depends upon itsThe susceptibility of an enzyme to proteolytic degradation depends upon its
conformation that may be altered by the presence or absence of substrates,
coenzymes, and metal ions.
Long-lived proteins, under physiological conditions, are
degraded at nearly constant rates, mostly nonselectively.
Nutritional deprivation (starving) increases selectively theNutritional deprivation (starving) increases selectively the
degradation rates of enzymes that can be missed and are not
necessary for survival of the cell.
6
necessary for survival of the cell.
2 Regulation of enzyme activity2 Regulation of enzyme activity
is a more rapid type of control than the control of enzyme synthesis.is a more rapid type of control than the control of enzyme synthesis.
The enzyme activities can be changed effectively in several ways:
– activation of proenzymes by partial proteolysis of the proenzyme,
– allosteric control and cooperative effects of enzymes that– allosteric control and cooperative effects of enzymes that
consist of several identical subunits,
– control arising from interactions with regulatory proteins– control arising from interactions with regulatory proteins
(e.g. activation of enzymes by releasing of inhibitory subunits
or another regulatory protein),or another regulatory protein),
– control by reversible covalent modification of enzymes or of
regulatory proteins; the most important example of this isregulatory proteins; the most important example of this is
reversible phosphorylation, catalyzed by protein kinases
and controlled by extracellular signals.
7
2 Regulation of enzyme activity
– Activation of an enzyme by partial proteolysis of the
proenzymeproenzyme
Active enzymes are formed from proenzymes molecules by
irreversible splitting of certain part(s) in their polypeptide chain.irreversible splitting of certain part(s) in their polypeptide chain.
This principle of activation is frequent among proteinases,
because it prevents against unwanted breakdown of proteins.because it prevents against unwanted breakdown of proteins.
Examples:
Extracellular – "big“ proteinases of the gastrointestinal tractExtracellular – "big“ proteinases of the gastrointestinal tract
(pepsin, chymotrypsin, trypsin, etc.),
– proteinases in the blood clotting cascade– proteinases in the blood clotting cascade
(coagulation factors IX, X, XI, and thrombin);
intracellular proteinases – activation of caspases that initiateintracellular proteinases – activation of caspases that initiate
apoptosis).
8
2 Regulation of enzyme activity
– Allosteric regulation of activity and cooperative effects– Allosteric regulation of activity and cooperative effects
Regulatory enzymes are frequently oligomers that consist of
several identical subunits (protomers). Their saturation curves
usually deviate from hyperbolic (Michaelis) shape, they are
sigmoid.
Cooperative effect – In these oligomeric enzymes (and also in some noncatalysts,
e.g. haemoglobin) the binding of substrates (or O to haemoglobin,
sigmoid.
catalysts, e.g. haemoglobin) the binding of substrates (or O2 to haemoglobin,
resp.) to one of the active sites can affect the affinity of active sites for
substrates in the other subunits. The effect becomes positively cooperative,
when it facilitates, due to induced changes in conformation, substrate binding to
the other subunits and so activates the enzyme.
Allosteric effectors are molecules that are allosteric to the
substrate (having structures distinct from the substrate) and
can bind reversibly to specific sites other than the enzymes´can bind reversibly to specific sites other than the enzymes´
active sites (to the allosteric sites). The induced change in
conformation results either in higher activity of the enzymes or in
9
conformation results either in higher activity of the enzymes or in
inhibition.
Regulation of allosteric enzymes – examples:
2 Regulation of enzyme activity
Regulation of allosteric enzymes – examples:
Cooperative effect
Allosteric enzyme
Cooperative effect
of the substrate Allosteric activator Allosteric inhibitor
Glycogen synthase – Glc-6-P -Glycogen synthase – Glc-6-P Glycogen
phosphorylase –
Glc-1-P, AMP Glc-6-P
Phosphofructokinase Fru-6-P citrate, ATP
Fru-1,6-bisphosphatase Fru-1,6-P2 Fru-2,6-P2
phosphoenolpyruvate
Fru-2,6-P2, ADP
Fru-1,6-bisphosphatase Fru-1,6-P2 Fru-2,6-P2
Pyruvate kinase phosphoenolpyruvate Fru-1,6-P2 alanine
phosphoenolpyruvate
Pyruvate dehydrogenase acetyl-CoA,
ATP, NADH
Isocitrate dehydrogenase ADP
––
–Isocitrate dehydrogenase ADP ATP, NADH
Pyruvate carboxylase acetyl-CoA citrate
–
–
10
–
2 Regulation of enzyme activity
– Control of enzyme activity by regulatory protein
Examples: Protein kinase A forms inactive tetramers C2R2. If two
regulatory subunits R bind four molecules cAMP, two catalytically active
– Control of enzyme activity by regulatory protein
regulatory subunits R bind four molecules cAMP, two catalytically active
subunits C are released. The decrease in cAMP concentration supports
interactions between C and R subunits, the inactive tetramer is restored.interactions between C and R subunits, the inactive tetramer is restored.
Phosphoprotein phosphatase 1 has a regulatory subunit, which keeps up
active complex of glycogen with the catalytic subunit.active complex of glycogen with the catalytic subunit.
If the regulatory unit is phosphorylated by PK A, it releases the catalytic
subunit (exhibiting low activity) that is then fully inactivated by binding withsubunit (exhibiting low activity) that is then fully inactivated by binding with
an similarly phosphorylated protein inhibitor. If it is phosphorylated at
another site by insulin-dependent PK, the phosphatase activity of the
complex of glycogen and the catalytic subunit will increase.
Proteinases often occur in the inactive forms, bound reversibly to the more
or less specific proteins (proteinase inhibitors). Plasma proteinase thrombin
is inactivated by binding to antithrombin, intracellular Ser- or Cysproteinases
are inhibited by various types of serpins and cystatins.
11
proteinases are inhibited by various types of serpins and cystatins.
2 Regulation of enzyme activity
– Reversible covalent modification of proteins:
– phosphorylation of proteins catalyzed by protein kinases (PK);
phosphate ester originates by the transfer of γ-phosphate from ATP,
dephosphorylation (hydrolysis) is catalyzeddephosphorylation (hydrolysis) is catalyzed
by phosphoprotein phosphatases;
– acetylation (e.g., of histones in nucleosomes),– acetylation (e.g., of histones in nucleosomes),
through transfer of acetyl from acetyl-CoA;
– ADP-ribosylation (e.g. GαS, EF-2, RNA polymerases),– ADP-ribosylation (e.g. GαS, EF-2, RNA polymerases),
transfer of ADP-ribosyl from NAD+, nicotinamide is released;
– myristoylation, farnesylation (prenylation), and many other.– myristoylation, farnesylation (prenylation), and many other.
γγγγ-Carboxylation of glutamyl residues side chains (prothrombin and other
factors in the blood-clotting cascade, osteocalcin, etc.) is obviouslyfactors in the blood-clotting cascade, osteocalcin, etc.) is obviously
irreversible, but it is important in formation of binding centres for Ca2+ ions,
essential for the biological activity of the protein.
12
essential for the biological activity of the protein.
Reversible phosphorylation of proteins
2 Regulation of enzyme activity
Reversible phosphorylation of proteins
is an intracellular reaction. ATP is the donor of phosphate.
protein kinasesPhosphorylation is catalyzed by highly specific protein kinases (PK).
Protein kinases are the largest family of homologous enzymes known –
there are more than 550 human types of protein kinases.
Proteins are phosphorylated either on serine or threonine residues
(alcoholic groups), or on residues of tyrosine (phenolic hydroxyl),
at specific positions within the polypeptide chains.
Activation of various protein kinases is specific – e.g. cAMP, cGMP,
Ca2+-calmodulin complex, etc. (see next table).
The signal that activates protein kinases is amplified (activation of one
enzyme molecule results in phosphorylation of numerous protein
molecules).molecules).
Dephosphorylation of phosphoproteins (hydrolysis of the
ester bond) is catalyzed by phosphoprotein phosphatases.
13
ester bond) is catalyzed by phosphoprotein phosphatases.
Examples of protein kinases (PKs):
2 Regulation of enzyme activity
Examples of protein kinases (PKs):
Phosphorylation of Ser/Thr residues Activated by
Protein kinases A cAMP
Protein kinases G cGMP
Protein kinases C diacylglycerol (and Ca2+)
AMP-dependent PK AMP
Ca2+/CaM-dependent PKs Ca2+ or Ca2+-calmodulin
PIP3-dependent PK-1 phosphoinositide 3,4,5-trisphosphate
Mitogen-activated PKs (MAP, MAPKK) growth factors, cellular stress
Cyclin-dependent PK cyclins (regulatory proteins)
Phosphorylation of tyrosine residues (tyrosine kinases)
– receptor types – e.g., insulin receptor or receptors of some growth– receptor types – e.g., insulin receptor or receptors of some growth
factors (IGF1,2, epidermal growth factor)
– intracellular, non-receptor types (e.g., Janus kinases) activated by
14
– intracellular, non-receptor types (e.g., Janus kinases) activated by
membrane receptors of growth hormone, prolactin, erythropoietin, cytokines.
2 Regulation of enzyme activity
Examples of regulation by reversible phosphorylation:
Activated by phosphorylation Inhibited by phosphorylationActivated by phosphorylation Inhibited by phosphorylation
glycogen phosphorylase-b-kinase
glycogen phosphorylase
glycogen synthase
glycogen phosphorylase
(glycogenolysis)
glycogen synthase
(glycogen synthesis)
fructose 2,6-bisphosphatase fructose 6-phosphate 2-kinasefructose 2,6-bisphosphatase
(gluconeogenesis)
fructose 6-phosphate 2-kinase
pyruvate dehydrogenase
(glycolysis)
acetyl-CoA carboxylase
(fatty acid synthesis)
HMG-CoA reductaseHMG-CoA reductase
(cholesterol synthesis)
15
3 Regulation of the transport across membranes3 Regulation of the transport across membranes
Examples:
– Insulin stimulates glycolysis, because it also promotes the uptake of
glucose by muscle and adipose tissue. Binding of insulin to its receptor
leads to a rapid increase in the number of GLUT4 transporters inleads to a rapid increase in the number of GLUT4 transporters in
the plasma membrane of rhabdomyocytes and adipocytes.
– The fatty acid synthesis and degradation are reciprocally regulated so
that both are not simultaneously active. Malonyl-CoA (present in cytosol
when there is a abundant supply of nutrients to the cell) inhibitswhen there is a abundant supply of nutrients to the cell) inhibits
carnitine acyltransferase I, thus preventing access of fatty acyl-CoAs
to the mitochondrial matrix and the enzymes that catalyze their oxidation.to the mitochondrial matrix and the enzymes that catalyze their oxidation.
On the contrary, fatty acyl-CoAs (present in cytosol at a high level in
fasting) inhibit the mitochondrial tricarboxylate transporter,fasting) inhibit the mitochondrial tricarboxylate transporter,
thus preventing activation of acetyl-CoA carboxylase by outflow of citrate
from mitochondrial matrix.
16
Mechanism of hormone and neurotransmitter actionMechanism of hormone and neurotransmitter action
Signal molecule types in neurohumoral regulations:
HORMONES secreted by endocrine glands, by dispersed
glandular cells (eicosanoids by many other
Signal molecule types in neurohumoral regulations:
glandular cells (eicosanoids by many other
cellular types);
NEUROHORMONES secreted by neurons into the blood circulation;NEUROHORMONES secreted by neurons into the blood circulation;
NEUROTRANSMITTERS secreted by neurons at nerve endings;
CYTOKINES secreted by immunocompetent cells;CYTOKINES secreted by immunocompetent cells;
GROWTH FACTORS secreted by various types of cells.
Signal molecules can be also classified as
- endocrine - carried by the blood, may act in the whole body,- endocrine - carried by the blood, may act in the whole body,
- paracrine - act within short distances of the site of their production,
- autocrine - act on the cells that produce them.
17
Hierarchical arrangement and signal amplificationHierarchical arrangement and signal amplification
of some regulatory processes
Secreted per day:Example:
Neurons of brain cortex NEUROTRANSMITTERS at most nanograms
Secreted per day:Example:
Neurons of brain cortex NEUROTRANSMITTERS at most nanograms
CORTICOLIBERIN micrograms
Specific neurons
in the hypothalamus
CORTICOLIBERIN micrograms
CORTICOTROPIN hundreds of micrograms
in the hypothalamus
Cells in the anterior lobe
of the pituitary CORTICOTROPIN hundreds of microgramsof the pituitary
Cells of the adrenal cortex CORTISOL tens of milligrams
CELLS IN THE TARGET TISSUES
18
Factors influencing the biological effects of hormones:
HORMONE SYNTHESIS CONTROL OF GENE
EXPRESSION
HORMONE STORAGE
FEED-BACK
CONTROL SECRETION SECRETION STIMULUS
specific
- e.g. liberin,
Metabolism
INACTIVATION
TRANSPORT
- e.g. liberin,
glandotropin
non-specific
- e.g. Ca2+, H+
INACTIVATION
TARGET CELL RECEPTOR
SIGNAL-TRANSDUCTION PATHWAY
- e.g. Ca , H
EXCRETION
SIGNAL-TRANSDUCTION PATHWAY
STATE OF THE
TARGET CELL
The hormone concentrations in blood plasma need not correlate with the biological effects!.
BIOLOGICAL EFFECT
19
The hormone concentrations in blood plasma need not correlate with the biological effects!.
TRANSDUCTION OF EXTRACELLULAR SIGNALS
The size and polarity of a signal molecule is decisive.
How cells receive, process, and respond to information from the environment?
The size and polarity of a signal molecule is decisive.
– Proteins and small polar signal molecules (amino acids, peptides,
biogenic amines, eicosanoids) don't penetrate across plasmabiogenic amines, eicosanoids) don't penetrate across plasma
membranes. They bind onto specific membrane receptors (integral
membrane proteins).
Binding of the ligand to the receptor results in a conformational change
of the intracellular domain, which either generates an increase of
intracellular concentration of a small secondary signal moleculeintracellular concentration of a small secondary signal molecule
(the second messenger), or directly activates a proteinkinase.
– Nonpolar signal molecules (steroids, iodothyronines, retinoates)– Nonpolar signal molecules (steroids, iodothyronines, retinoates)
diffuse through the plasma membranes of all cells and bind to specific
proteins - intracellular receptors.proteins - intracellular receptors.
Complexes hormone-receptor then enter the nuclei, binds to a specific
region of DNA (hormone response element, HRE),
and activate (or repress) gene transcription.
20
and activate (or repress) gene transcription.
Membrane and intracellular receptors
Nonpolar signal moleculePolar signal molecule
Membrane and intracellular receptors
Nonpolar signal moleculePolar signal molecule
Membrane receptorMembrane receptor
diffusion
Intracellular receptor
Signal transduction
Interaction of the complex
Intracellular receptor
Amplification
Interaction of the complex
hormone-receptor with DNA
Biological response
Biological response
(the effect is slow, either early or late)
Biological response
(prompt effect)
21
(the effect is slow, either early or late)
Main types of membrane receptors
Receptors – ion-channels (ROC, ligand gated ionophores) serve
exclusively as receptors for neurotransmitters (see lecture 7).exclusively as receptors for neurotransmitters (see lecture 7).
Receptors activating G-proteins (heterotrimeric G-proteins), the result
of specific ligand binding is mostlyof specific ligand binding is mostly
- stimulation or inhibition of adenylate cyclase,
- stimulation of phospholipase C,
- stimulation of phosphodiesterase.- stimulation of phosphodiesterase.
Receptors exhibiting intrinsic catalytic activity
- guanylate cyclase activity – receptors for natriuretic peptides,- guanylate cyclase activity – receptors for natriuretic peptides,
- tyrosine kinase activity
- insulin receptor, receptors for insulin-li growth factors (IGF1,2),- insulin receptor, receptors for insulin-li growth factors (IGF1,2),
- dimerizing receptor for epidermal growth factor (EGF).
Receptors cooperating with non-receptor tyrosine kinasesReceptors cooperating with non-receptor tyrosine kinases
(e.g., Janus kinase, JAK) – receptors for somatotropin (growth
hormone), prolactin, erythropoietin, interferons, interleukins and other
cytokines.
22
cytokines.
Family of heterotrimeric G-protein-coupled receptors
All receptors of this type exhibit common structural features:
Family of heterotrimeric G-protein-coupled receptors
All receptors of this type exhibit common structural features:
Extracellular parts (the N-end and
hydrophilic loops) are slightly glycosylated;hydrophilic loops) are slightly glycosylated;
α-helical segments IV, VI, and VII form a
"pocket", the specific binding site for the
agonist. There are also accessory binding
H2N
agonist. There are also accessory binding
sites for antagonists.
Seven αααα-helical segments span theSeven αααα-helical segments span the
membrane and are connected by intra- and
extracellular hydrophilic and more divergent
loops.loops.
Intracellular domains represent the binding
-COOH
Intracellular domains represent the binding
site for the specific G-protein type.
23
G-proteins
are GTP- and/or GDP-binding proteins, mostly freely membrane-boundare GTP- and/or GDP-binding proteins, mostly freely membrane-bound
(they can move along the inner surface of the plasma membrane).
G-proteins participate in various types of the second messengerG-proteins participate in various types of the second messenger
production.
All types of those G-proteins have a similar structure and mechanism
of activation.of activation.
Heterotrimers consist of subunits αααα, ββββ, and γγγγ.Heterotrimers consist of subunits αααα, ββββ, and γγγγ.
Gββββ and Gγγγγ subunits are hydrophobic and nonspecific,
Gαααα subunit is the largest, hydrophilic, it binds GTP or GDP, andGαααα subunit is the largest, hydrophilic, it binds GTP or GDP, and
is specific for particular mechanism of second messenger production.
More than 20 different α subunits have been identified.More than 20 different α subunits have been identified.
Examples – see table (picture number 26).
24
The cycle of G-proteins activation
Complex
receptor-specific ligand GTP
The cycle of G-proteins activation
Complex
receptor-specific ligand
Complex receptor-ligand-trimer
Gα-GDP,Gβ,Gγ
GTP
Trimer Gα-GDP,Gβ,Gγ
-trimer Gα-GDP,Gβ,Gγ
Dimer Gβ,Gγ
GDP
Dimer Gβ,Gγ
Activated
subunit Gα-GTP
InactiveDimer Gβ,Gγ Activated
subunit Gα-GTP
Inactive
subunit Gα-GDP
Dimer Gβ,Gγ
Interaction
with the target protein
Pi
PRODUCTION OF THE
SECOND MESSENGER
25
ααααSelected types of G protein αααα-subunits
Examples of Effect of activated GExamples of
activating receptors
Effect of activated Gα
on the target protein
Gα subunit type
Gαααα s (s for stimulatory)
glucagon,
parathyrin,
β-adrenergic
stimulation of
adenylate cyclaseβ-adrenergic adenylate cyclase
somatostatin,
α -adrenergic
(i for inhibitory)Gαααα i
inhibition of
adenylate cyclaseα2-adrenergic
vasopressin V1,
endothelin ET ,
(activating the PI cascade)Gαααα q
adenylate cyclase
stimulation ofendothelin ETA,B,
acetylcholine M1
α1-adrenergic
(activating the PI cascade)Gαααα q
(t for transducin)G
stimulation of
phospholipase C
rhodopsin(t for transducin)Gαααα t stimulation of
cGMP phosphodiesterase
26
Hormone receptors that activate Gs or Gi proteins
stimulates or inhibit adenylate cyclasestimulates or inhibit adenylate cyclase
Adenylate cyclase, a membrane-bound enzyme, catalyzes the reactionAdenylate cyclase, a membrane-bound enzyme, catalyzes the reaction
ATP →→→→ cAMP + PPi ; the second messenger is cyclic AMP.
receptor receptor
ligand ligand
AMP-cyclasereceptor receptor
GααααS Gααααi
βγβγβγβγββββγγγγ
ATP
cAMP
phosphodiesterase
proteinkinase A
inactive (R2C2)
AMP H2O
phosphodiesterase
active proteinkinase A
2 C + 2 R(cAMP)2
phosphorylations
27
2 C + 2 R(cAMP)2
Receptors that activate Gq protein stimulateReceptors that activate Gq protein stimulate
phospholipase C and start the phosphatidylinositol cascade
Phospholipase C catalyzes hydrolysis of phosphodiester bond in
phosphatidylinositol 4,5-bisphosphate to
diacylglycerol and inositol 1,4,5- trisphosphate:diacylglycerol and inositol 1,4,5- trisphosphate:
Both reaction products are the second messengers:Both reaction products are the second messengers:
Inositol 1,4,5-trisphosphate opens the Ca2+ channel in ER membrane,
diacylglycerol activates proteinkinase C.
28
diacylglycerol activates proteinkinase C.
Phosphatidylinositol cascadePhosphatidylinositol cascade
specific ligand activation of
phospholipase Creceptor
specific ligand
PIP2 DG
activation of
proteinkinase C
phospholipase Creceptor
Gααααq
βγβγβγβγ
PIP2 DG
IP3 active proteinkinase C
increase of [Ca2+]
in cytoplasm
phosphorylations
in cytoplasm
Endoplasmic reticulumEndoplasmic reticulum
Ca2+
IP receptors in the membranes of ERIP3 receptors in the membranes of ER
act as ligand gated channels for Ca2+ ions
29
Receptors having intrinsic catalytic activities
Insulin receptors has an intrinsic tyrosine kinase activity
β
α
-S-S-
-S-S-
α
-S-Sof
the intracellular domains of β subunits.
β
-S-S-
β
Binding of insulin to its specific receptor stimulates autophosphorylation
of β subunits and phosphorylation of IRS 1-4 (insulin receptor substrates 1-4).
Insulin
α
-S-S-
-S-S-
α
-S-S-
β
-S-S-
-S-S-
β
-S-S-P-P
P-Pactivation
and docking of
PI-3-kinase on membrane-P-P P-P-
IRS1-4
-PIRS1-4
PI-3-kinase on membrane
activation of phosphoprotein phosphatase-1
activation of small G-protein Ras
30
activation of small G-protein Ras
Insulin receptor substrates 1-4 are adaptor proteins.
If phosphorylated by the insulin-receptor complex, they bind toIf phosphorylated by the insulin-receptor complex, they bind to
other proteins that are activated in this way.
Among others,
– the lipid kinase PIP2 3-kinase is activated. The product PIP3 initiates
activation of the kinase PDK-1 (PIP -dependent kinase) which, in turn,
Among others,
activation of the kinase PDK-1 (PIP3-dependent kinase) which, in turn,
activates protein kinase PK B. The consequence is
exposition of transporters GLUT4 into membranes of skeletal musclesexposition of transporters GLUT4 into membranes of skeletal muscles
and adipocytes.
– Regulatory subunit of phosphoprotein phosphatase-1 is activated
resulting in activation of its phosphatase activity which
dephosphorylates both glycogen synthase and phosphorylase.
– Phosphorylation of IRS also results in docking of proteins Grb2 and SoS– Phosphorylation of IRS also results in docking of proteins Grb2 and SoS
and activation of small G-protein Ras which triggers, through binding
onto protein kinase Raf, the cascade of phosphorylations called theonto protein kinase Raf, the cascade of phosphorylations called the
Ras signalling pathway (mitogen-activated protein kinases, MAPKs)
important in the regulation of proliferation and differentiation of several
cell types.
31
cell types.
Dimerizing receptor for EGF (epidermal growth factor)
containing an intrinsic tyrosine kinase activity
2 EGF molecules
R R
2 EGF molecules
R R
R R
-P
-PPP-
SoS
Ras–GTP
Raf phosphorylation
Autophosphorylation of the receptor enables linking of proteins Grb2
-PP- SoS Raf
phosphorylation
cascade
phosphorylation
Autophosphorylation of the receptor enables linking of proteins Grb2
and SoS which bind and so activate the Ras signalling pathway.
The small G-protein Ras (Ras-GDP) after an exchange of GDP for GTPThe small G-protein Ras (Ras-GDP) after an exchange of GDP for GTP
activates the serine protein kinase Raf and initiates the phosphorylation
cascade catalyzed by protein kinases MAPKs (mitogen-activated PKs)
and ERKs (extracellular signal-regulated PKs).
The consequence is phosphorylation of transcription factors and regulation
of gene expression.
32
of gene expression.
Receptors activating non-receptor tyrosine kinases
Dimerizing receptors activating tyrosine kinases JAK
(Janus kinases) – e.g., receptors for prolactin, growth hormone,(Janus kinases) – e.g., receptors for prolactin, growth hormone,
erythropoietin, interferon, various interleukins and other cytokines.
ligand
dimerizationdimerization
–PSTAT
STATSTAT
dimerization of STAT
and nuclear translocation
(signal transducers and
activators of transcription)
Upon ligand binding, these receptors dimerize and interact with a cytosolic
tyrosine kinase JAK which is autophosphorylated and phosphorylates the
receptor on tyrosine residues. The STAT proteins (signal transducers and
activators of transcription) associate with the receptor and areactivators 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.
33In a similar way, phosphorylated receptors activate MAP kinase cascade.
Intracellular receptors
The general features of the function of all these receptors are very
Intracellular receptors
of steroid hormones (and calcitriols), iodothyronines, and retinoates
The general features of the function of all these receptors are very
similar. The hormone-receptor complexes binds to specific regions of
DNA (called hormone response elements, HRE) and
activate or inactivate transcription of specific genes.activate or inactivate transcription of specific genes.
hydrophobic hormone molecule
(cortisol) diffuses into the cell(cortisol) diffuses into the cell
active complex receptor-ligand (monomer),active complex receptor-ligand (monomer),
hsp 90 and other proteins are released
inactive receptor
active complexes form dimers and
are translocated into the nucleusinactive receptor
for glucocorticoids (GR)
forms in the cytoplasm
complex with hsp 90 dimer
are translocated into the nucleus
through nuclear pores
34
complex with hsp 90 dimer
and other proteins
Glucocorticoid receptor (GR) – functionGlucocorticoid receptor (GR) – function
DNA
GRE
DNA binding domain
GR
cortisol binding domainDNA binding domain cortisol binding domain
(hydrophobic pocket)
Active complex cortisol-receptor binds onto DNA at the specific sequence
GRE (glucocorticoid response element, quite generally HRE – hormoneGRE (glucocorticoid response element, quite generally HRE – hormone
response element), after the coactivators and specific
hormone response element-binding proteins (HREB-proteins) has
been attached. So the complex acquires the ability to act as enhancer thatbeen attached. So the complex acquires the ability to act as enhancer that
supports initiation of transcription on the promoter..
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Active complex cortisol-receptor binds onto DNA at the specific sequence
Initiation of transcription by cortisol
Active complex cortisol-receptor binds onto DNA at the specific sequence
GRE (glucocorticoid response element, one of the HRE – hormone response
elements).
The coactivator and specific hormone response element-binding proteins
(GREB-proteins) are also attached. This complex acquires the ability to act as
enhancer that supports initiation of transcription on the promoter by means of
cortisol-GR dimer complex
enhancer that supports initiation of transcription on the promoter by means of
mediator proteins.
enhancer
coactivatorGREB protein
GRE
cortisol-GR dimer complex
CTD
> 1 000 bp
mediator proteins
coactivatorGREB protein
basal
TF IID Pol II
CTD
promoter
basal
transcription
apparatus
GR dimer – intracellular glucocorticoid receptor (dimer)
GRE – glucocorticoid response element
promoter
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GREB protein – GRE binding protein (a specific transcription factor)