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BIOCHEMISTRY
GENERAL MEDICINE
HORMONAL REGULATION
RNDr. Zdeněk DVOŘÁK, PhD.Department of Medical Chemistry and BiochemistryFaculty of Medicine, Palacky University Olomouc
HORMONAL REGULATION
Compounds involved in the co-ordination of metabolic activities of various organsand tissues – cellular signalling ; signal transduction
• HORMONES• NEUROTRANSMITTERS• GROWTH FACTORS• CYTOKINES
• Extracellular signalling substances• Synthesis in one class of cell
transmission
TARGET CELLS
HORMONES• Synthetized by specific tissues – ENDOCRINE GLANDS• Secreted directly into the BLOODSTREAM and carried to the ir sites of action• Specifically alter METABOLIC ACTIVITIES of TARGET CEL LS (remotefrom secretory organ)• Active at very low concentrations (pM – µµµµM)• Rapidly metabolized – SHORT-LIVED EFFECT
SIGNAL TRANSDUCTION
SIGNAL
ULTIMATECELLULARRESPONSE
HORMONE RECEPTOR
TRANSDUCTOR
EFFECTOR
SECOND MESSENGER
INTEGRATION AND CONTROLOF METABOLIC PROCESSES
Senzory inputs from the environment
CENTRAL NERVOUS SYSTEM
Hypothalamus
Anterior pituitary Posterior pituitary
Primary target
OxytocinVasopresinProlactinFolicule
Stimulatinghormone
Luteinizinghormone
SomatotropinCorticotropinThyrotropin
Secondary target
Thyroid Adrenalcortex
PancreaticIslet cells
Adrenalmedula
Ovary Testis
TestosteroneProgesterone
EstradiolInsulin
GlucagonSomatostatin
CortisolCorticosterone
Aldosterone
ThyroxineTriiodo-thyronne
EpinephrineNor-
epinephrine
MusclesLiver
Manytissues
LiverMuscles
Reproductive organsLiverMusclesHeart
Mammaryglands
SmoothMuscle;
Mammaryglands
Arterioles
Ultimate target
CLASSIFICATION OF HORMONES ACCORDING TO STRUCTURE
1. Derived from aminoacids - epinephrine (adrenaline)- nor-epinephrine- thyroxine- triiodthyronine epinephrine
2. Peptides and aminoacids - insulin; glucagon- liberins; oxytocin; vasopresin- ADH; ACTH
oxytocin
3. Steroids - cortisol; aldosterone- progesterone; estradiol; testosterone
estradiol4. Eicosanoids - prostaglandins; leucotrienes
- prostacyclines; thromboxanes
arachidonic acid
CLASSIFICATION OF HORMONES ACCORDING TO MODE OF ACTION
1. Hormone DOES NOT TRAVERSE plasma membrane of a targ et cell• amino acids; peptides• hydrophylic compounds• binding of hormone to the RECEPTOR at the CELL SURFACE• hormonal response inside the cell – changes in AFFINITY of key proteins/enzymes• ACTION THROUGH: - second messengers
- activation of enzyme activity of cytosolic receptor domain- opening ion channels
• SHORT-TERM action; ULTRA-RAPID response
2. Hormone TRAVERSES plasma membrane of a target cell• steroid and thyroid hormones• lipophylic compounds• binding of hormone to the INTRACELLULAR RECEPTOR• HORMONE-RECEPTOR complex binds to DNA and triggers TRASCRIPTION ofspecific genes – changes in LEVEL of key proteins/enzymes• LONGTERM action; SLOW response
RECEPTORS
• proteins• located in plasma membrane or in interior of the cell
• 2 binding sites - hormone binding site- a component of signal transduction system
HORMONE BINDING(saturable; M.-M. kinetics)
Extensive conformationchange of receptor
ACTIVATION OF SECONDBINDING SITE
GRGR
GRE
PP
GRGR
TR RAR
Peptides, AA
steroids
retinoids
thyroid
HORMONE
Several classesof receptor
Identical hormonebinding site
Different secondbinding site
Various physiological effects(different tissue)
ADRENERGIC RECEPTORSαααα1 – coupled to phosphatidyl inositol cascadeαααα2, ββββ1, ββββ2 – coupled to adenylate cyclase cascade
αααα1 – salivary gland ↑ K+; ↑ H2O secretionαααα2 – pancreatic β−cells ↓ secretion
- muscle ↑ glycogenolysisββββ1 – heart ↑ rate; contraction force
- adipocyte ↑ lipolysisββββ2 – liver ↑ glycogenolysis
• increase in receptor density in the cell = increased ce llular response
GTP- binding proteins = G - proteins• guanyl-nucleotides proteins• TRANSDUCTORS = carriers of excitation signal from RECEPTOR to EFFECTORwithin plasma membrane
• peripheral membrane proteins (located on cytosolic side)• TRIMER = it consists of αααα, ββββ and γγγγ subunits• αααα subunit is binding site for GDP/GDP; GTPase activity
• large extracellular ligand binding domain• formation of hormone-receptor complex• conformation change of the receptor • transduction of signal inside the cell
• receptor interacts with G-protein• exchange of GDP for GTP in αααα-subunit
• release of αααα-(GTP) subunit • activation of effector
• hydrolysis of GTP to GDP • release of αααα-GDP from effector• re-association with ββββ and γγγγ subunits
• Inhibition of GTPase activity of αααα-GTP subunit results in irreversibile inactivationof effector and consequently in uncontrolled cellular res ponse
• Example: Vibrio cholerae toxin inhibits GTPase activity in enterocytes that inturn leads to permanent activation of the effector (adeny late cyclase). As theresult; the secretion of Na+ and H 2O is uncontrolled and severe dehydratation oforganism is developed.
G - protein signalling
G - proteins
GS - stimulatory
Gi - inhibitory
Interaction withdifferent receptors
Stimulus Receptor G-prot. Effector Response
Epinephrine β-adrenergic r. Gs Adenylate cyclase Glycogen breakdownSerotonin Serotonin r. Gs Adenylate cyclase Behavioral sensitizationLight Rhodopsin Transducin cGMP phosphodiest. Visual excitationIgE-antigen complex Mast cell IgE r. GPLC Phospholipase C Secretionf-Met peptide Chemotactic r. GPLC Phospholipase C ChemotaxisAcetylcholine Muscarinic r. Gk Potassium channel Slowing pacemaker activity
SECOND MESSENGERS• amplified intracellular signals – products of activated effector action• small molecules or ions – allosteric effectors
• cAMP - cyclic adenosine monophosphate• cGMP - cyclic guanosine monophosphate• DAG - 1,2-diacylglycerol• IP3 - inositol-1,4,5-triphosphate• Ca2+ - calcium (free or bound to calmodulin)
SIGNAL TRANSDUCTION SYSTEMS
• ADENYLATE CYCLASE system• PHOSPHATIDYLINOSITOL system• TYROSINE KINASE system• GUANYLATE CYCLASE system• STEROID/THYROID/RETINOID SIGNALLING
ADENYLATE CYCLASE (AC) SYSTEM
• AC is activated via G-protein• effector = membrane AC
• AC converts ATP to cAMP• elevation of cytosolic cAMP• cAMP is second messenger
cAMP action• cAMP is allosteric activator of proteinkinases• activated proteinkinases phosphorylate target proteins• phosphorylated proteins (enzymes) have altered function s – cellular response
• ↑ degradation of storage fuels• ↑ HCl secretion by gastric mucosa• ↓ aggregation of blood platelets
ACTIVATION OF PROTEIN KINASE A (PKA) BY cAMP
• heterotetramer dissociates• active catalytic subunits of PKA are released
• PKA - 2 catalytic and 2 regulatory subunits• cAMP binds to regulatory subunits
• PKA phosphorylates cytosolic proteins• PKA may enter nucleus and phosphorylate TFs
• phosphorylated TFs recruit co-activators• gene expression is triggered
Hormones using cAMP as second messenger
• Calcitonin• Chorionic gonadotropin• Corticotropin• Epinephrine• Follicle-stimulating hormone• Glucagon• Luteinizing hormone• Nor-epinephrine• Lipotropin• Melanocyte-stimulating hormone• Parathyroid hormone• Thyroid-stimulating hormone• Vasopressin
cAMP signalling
GUANYLATE CYCLASE SYSTEMExtracellularside
Cytosolicside
PP
P
P
Peptide hormone(natriuretic factor)
PP
P
P
• cellular response• e.g. NO synthesis
GTP
cGMP
P
hydrolysis byphosphodiesterases
• inactivation• loss of cellular response
• inhibited by VIAGRA• prolonged cell response
PHOSPHATIDYL INOSITOL PHOSPHATE (PIP) SYSTEM
• principal molecule in the signalling isphosphatidyl inositol-4,5-bis-phosphate (PIP2)
• membrane receptor trasmits signaland activate effector – PI3 kinase
• PI3K phosphorylates PIP2 to PIP3 • phosphatidyl inositol-3,4,5-tris-phosphate (PIP3)
• PIP3 recruits tyrosin kinase (BTK) andphospholipase C (PLC)
• BTK phosphorylates PLC
• activated PLC cleaves PIP2 to 2 fragments• 2 second messengers!!!
• IP3 = inositol-1,4,5-triphophate• DAG = 1,2-diacylglycerol
• IP3 binds calcium channel in ER • channels open and releases Ca 2+ in cytosol
•Ca2+ binds protein kinase C (PKC)• PKC then goes to plasma membrane
•PKC is activated when both, i.e.calcium and DAG are bound
• PKC phosporylates its target proteins
PIP
Protein Kinase C
Important role in controlof cell division and proliferation
Phosphorylates variety of targetproteins; e.g.: insulin receptor;glucose carrier; CYP P450;tyrosine hydroxylase etc.
• PIP system is activated via variety of stimuli; e.g. AC TH, epinephrine,Neurotransmitters, growth factors, antigens• Inactivation of PIP system: IP3 IP2
• PIP system mediates variety of effects; e.g. Glycogeno lysis in liver cells;Histamine secretion by mast cells; Serotonine release by blood platelets;Insulin secretion by pancreatic islet cells; Smoth musc le contraction;Epinephrine secretion by adrenal chromaffin cells; visu al transduction
calmodulin
Ca2+
Endoplasmicreticulum
Ca2+
Ca2+
Ca2+ is released from ERin response to hormonesor neurotransmitters
Ca2+
calmodulinCa2+
Ca2+
Ca2+
Ca2+
ComplexCalmodulin-Ca2+
Transinet increase ofintracellular Ca2+ favorsformation of complex
inactiveenzyme
calmodulinCa2+
Ca2+
Ca2+
Ca2+active
enzyme
substrate product
Calmodulin-Ca2+ complexis an essential componentof many Ca2+ dependentenzymes
TYROSINE KINASE (TK) SYSTEM
• membrane receptor – contains effector domain• effector = tyrosine kinase (TK); protein kinase specifi c for phosphorylationof tyrosine residue• second messenger = O ? x IRS• extracellular signals – insulin, nerve growth factor; e pidermal growth factor
insulin
P
P
P
autophosphorylation
ATP ADP
• catalytic activity of TK switched onby insuline binding • enhanced activity independent
of insuline binding
TYROSINE KINASE (TK) SYSTEM
• hormone binds TK = H-R complex = TK activation
• TK phosphorylates target proteins (e.g. phosphatases; amino acidtransporter; glucose transporter etc.) = CELLULAR RES PONSE
• autophosphorylation of TK ensures that TK activity is swi tched on even inabsence of hormone = long term effect of insulin
• insulin has general growth-promoting properties – it act s as metabolic activatorand growth factor almost in all cells in the body it acts
• SHORT-TERM effects = polysaccharide and fat synthesis• LONG-TERM effects = nucleic acud and protein synthesis
1
1
2
2
3
3
4
4
5
5
membrane receptors
OUTSIDE CELL
Hormonalsignals
INSIDECELL
Cyclic AMP Cyclic GMP Ca2+ DAG Proteins
G-prot G-prot G-prot G-prot Tyrosinkinase
secondmessengers
Nitricoxide
IP3
PKA PKG Calmodulin PKC Ser/Thr kinases
Dedicatedkinases
Multifunctionalkinases
Protein substrates Protein substrates
Protein substratesOther phospholipases
• steroid/thyroid receptors – intracellular localization• cytosolic - glucocorticoid; estrogen; androgen; progesterone; mineralocorticoid(undergo nucle-cytosolic traslocation)• nuclear – retinoid (RARs, RXRs), vitamine D, thyroid
• no second messenger
• DNA is the effector
• binding to DNA – triggering gene expression = cellular response• enhanced synthesis of specific proteins
STEROID AND THYROID HORMONES ACTION
Hormone
Receptor
+
Hormonetraversplasma
membrane
Outsideof cell
cytosol ornucleus
Nucleartranslocation
HR complexbinding to DNA
cellular response
Plasma-boundsteroid hormoneFree steroid hormone
HR
HR
HR HR
hsp
HR
HR
DNA
HRtranscription
mRNAmRNA
translation
Proteinsynthesis
NEW PROTEIN
Biological response
TR, VDR and RARs regulation of transcription : Hormone receptor (HR) is dimerizedand bound to DNA at hormone response element site. Without the ligand, transcriptionis inactive due to the interaction of HR with co-repressor. When hormone binds to HR,the bound co-repressor dissociates leading to an interaction between co-activator andHR. These regulatory changes result in increased transcription.
HR HR
ligand
DNA
RNA transcriptionCo-repressor
Co-activator
TranscriptionIniciationkomplex
Hormone response element
PROMOTER REGION
Hormone class Target organ Target gene
Glucocorticoids Liver tyrosine aminotransferasetryptophan oxygenaseα-fetoprotein (down-regulation)metallothionein
Liver, Retina glutamine synthetaseKidney Phophoenolpyruvate carboxykinase (PEPCK)Oviduct OvalbuminPituitary Pro-opiomelanocortin
Estrogens Oviduct Ovalbumin; Lysozyme
Progesterone Oviduct Ovalbumin; OvidinUterus Uteroglobine
Androgens Prostate AldolaseKidney β-glucuronidaseOviduct Albumin
1,25-dihydroxyvimin D3 Intestine Calcium-binding protein
Thyroid hormones Liver Carbamoyl phosphyte synthetase; Malic enzymePituitary Growth hormone; Prolactin (down-regulation)
Target organs and genes for steroid and thyroid hormones
HORMONE ACTION AND CARCINOGENESIS
Alteration of structure of any component in signal transducti on system
Loss of metabolic control in a cell
Transformation of normal cell
• G-proteins• insulin receptor• thyroid/steroid hormone receptors
• DAG
Analogs encoded by specific VIRALONCOGENES = proteins which bindhormone (HR complex) but LACKSWITCH OFF mechanism (lack of GTPaseactivity; permanent TK activity; tightbinding of hormone
Analogs (PHORBOL ESTERS) = causeactivation of protein kinase C = stimulationof tumor formation (mainly in presenceof carcinogen)
• Loss of metabolic control; uncontrolled cell growth; trans formationof a normal cell to a cancer cell
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