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

Obrázek IX/5

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