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Mechanisms of hormone actionHormones are chemical messengers synthesized by organisms that initiate biological responses by binding with high affinity and specificity to target cell receptors within the same individual

Endogenous substanceHigh affinity and specificity of binding to specific receptors on target cellsInitiates biological response

Function of hormones

HOMEOSTASISReproductionGrowth and developmentMaintenance of internal environmentProduction, utilization and storage of energy

Life of a hormone

Hormone transport protein

Chemical nature of hormones

Can be divided into 3 groups

Amino acid derivativesPeptide hormonesLipid derivatives

Amino acid derivatives

Derivatives of tyrosineCatecholamines (epinephrine, dopamine)Thyroid hormones (dipeptides)

Tryptophan derivativeMelatonin

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Peptide hormones

Glycoproteins from anterior pituitarythyroid-stimulating hormone (TSH) luteinizing hormone (LH) follicle-stimulating hormone (FSH)

Peptides and small proteinsDigestive tract hormonesPituitary hormonesPancreatic hormones

2 classes of lipid derived hormones

Steroid hormones: derived from cholesterol2 groups

with the intact steroid ring (adrenal and gonadalsteroids)with the steroid ring cleaved (metabolites of vit D)

Eicosanoids: derived from arachidonic acid

Catecholamines

Molecules with catechol groupHormonal regulators

Dopamine in hypothalamus inhibits prolactin secretionEpinephrine (adrenaline) – stress reaction

Synthesized from aa phenylalanine or tyrosine in enzymatic reactions

Synthesis of catecholamines

Synthesized in cytosolPackaged in vesicles and exocytosedWater soluble, do not need transport proteins Work from the outside from the cell (bind to surface receptors)

Synthesis of melatonin (indoleamine)

Tryptophan to serotoninNAT (N-acetyltransferase) is activated only in the darkWorks on surface receptors

Peptide hormone synthesis

Synthesized on the ribosomes attached to rough ER All of them have ER targeting sequence on the N-terminus (signal peptide)

Synthesized as prohormonesInactive molecules converted to active hormones in ER and Golgi, sometimes after secretion

Exocytosed from the cellWork from the outside from the cell (bind to surface receptors)

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Splice variantsAlternative processing of mRNA can result in splice variants of the same hormone

Availability of transcription factors can affect hormone production

Cell biology vs. endocrinology

Preprohormone – full sequence of the peptideProhormone – peptide minus signal sequence Can (and usually does) undergoes additional proteolyticcleavage in Golgi

Hormone convertaseHormone – biologically active product

Posttranslational processing

Signal peptide removalFolding (ER)

Formation of disulfide bondsGlycosylation (ER)

Posttranslational processing

Clevage (Golgi)Sometimes multiple copies or even different hormones are produced from the same prehormone

ProTRH 6 repeats in humans 5 repeats in rats

Peptide homology

Neurohypophyseal hormones

Peptide homology

Glycoproteins of anterior pituitaryAlpha subunit identical in all 3 TSH, LH and FSH

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Shape matters

Insulin and IGF (insulin like growth factor), a “real”growth hormone

Peptide hormone transport

Usually water soluble Transported in plasma - require no specific carrier mechanisms

Signaling process

Recognition of signalReceptors

TransductionChange of external signal into intracellular message

EffectModification of cell behavior

Hormone receptors

Molecules within or on the surface of target cells that bind hormones with high affinity and specificity and thereby initiate and mediate biological responsesHormones will only produce the response in cells that express the receptors for this particular hormone (target cells)

ONLY target cells respond to hormoneCells that do not have receptors for the hormone “ignore” the hormone

Properties of the hormone-receptor interactions

Tissue specificity - each organ has a unique set of hormone receptors

Peptide and amine receptors

Surface receptors a.k.a transmembrane receptorsPeptides and amines cannot cross the membrane

When activated, a receptor on the surface “passes” the signal to intracellular second messengers or directly to cellular effectors to produce biological response

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Many families of cell surface receptors

Based on homology and signaling strategyThe same ligand can bind to two or more different families!!!Multiple splice variants (β1 and β2 adrenergic receptors) can be tissue specific

G protein coupled receptors

Use G-proteins as molecular switch to turn on enzymes producing intracellular second messengers

G protein coupled receptors = GPCR

Ligand binding to the receptor activates a signal transduction cascade that comprises

G protein – molecular switchEnzyme that produces second messengersSecond messengersTarget protein - effector

But not necessary all steps are involved!!!!

Molecular properties of G protein coupled receptors

A.k.a. serpentine receptorsSeven transmembrane regions of 22-24 hydrophobic residues

N-terminus faces outside (ligand binding domain)C-terminus faces cytosolA cytosolic loop between helices 5 and 6 is the place for interaction with G protein

G proteins

Membrane bound heterotrimeric proteins consisting of 3 subunits α, β, γCoupled to surface receptorsMolecular switches

Use the exchange and hydrolysis of nucleotides (GTP/GDP) to transduce the signal from the surface receptors to intracellular effectors

G protein cycle

When G protein is inactive it is bound to GDP and exists as a trimerThe exchange of GDP for GTP activates G proteinG protein dissociates into two subunits: α and βγ dimer

GTP is bound to α subunitα Subunit has an intrinsic GTPase activity and hydrolyses GTP to GDP This process terminates the signalα and βγ reassociate

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What the G proteins can do?

Activate enzymes to produce second messengersActivate transcription factorsModulate ion channels, pumps and exchangersAffect cytoskeletonModulate enzymes

Adrenaline signaling

Amplification of signal by second messengers

Differential regulation of adenylatecyclase

Activated by GsInhibited by Gi

Activated CREB binds to CRE sequence and stimulates transcription

CREB needs to be phosphorylated at serine 133

Only genes that have CRE sequence are activated by those receptors

Regulation of transcription by cAMPkinase

CREB links cAMP signals to transcription

Only genes that have CRE sequence in front of them are activated by these receptorsCREB needs to be phosphorylated at serine 133Interacts with a co-activator CBP/P300Activated CREB binds to CRE sequenceCBP/P300 links CREB to transcription factorsand stimulates transcription

Second messengers

cAMP is not the only second messenger initiated by GPCRsIP3 (inositol 1,4,5 – trisphosphate) and DAG (diacylglycerol), are the second messengers for G proteins from the Gq family

They are made by phospholipase C (PLC) that breaks phoshatidylinositol 4,5 bisphosphate (PIP2) to IP3and DAG

Several other second messenger are derived from membrane lipids

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PIP2 breakdown

IP3 increases intracellular calcium levels via the release from intracellular storesDAG activates protein kinase C (PKC)

IP3 as a second messenger

Calcium is also intracellular second messenger

Regulation of hormone secretionRegulation of transcription through Ca- calmodulinkinase

Protein kinase C (PKC) signaling

Serine/threonine kinaseActivated by DAGPhosphorylates various cellular effectorsActivates transcription factors AP-1 (c-fos and c-jun are both protooncogenes)

Other lipid messengers Ceramide signaling

Product of sphingomyelin cycleSphingomyelins do not have glycerol backbone

Second messenger in TNF-α signaling and stimulation of apoptosisIncrease in prostaglandin biosynthesisActivation of transcription factor NF κb

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Catalytic receptors with intrinsic enzymatic activity

Ligand binding causes activation of enzymatic activity of the receptor (receptor itself is an enzyme)

Tyrosine kinaseGuanylyl cyclasePhosphatase

Modification of cellular activity

Receptor tyrosine kinases

Ligand binding causes dimerization of the receptorThis activates enzymatic activity of kinase domain and phosphorylation of the other subunitPhosphorylated tyrosine is recognized by molecules with SH2 domain that will propagate the signal to other cellular effectors

RTKs

Most RTK are monomers when not crosslinked by ligandsInsulin receptor stays as a dimer but ligand binding is necessary for phosphorylation

Signaling by RTK

Activation of enzymesActivation of Mitogen Activated Protein Kinase pathways (MAPK pathways)

Enzymes activated by RTKHow do RTKs activate MAPK pathways and affect transcription?Phosphotyrosine residues on the kinase interact with adapter proteinsTransmit a signal to Ras, a monomeric G protein (molecular switch)Ras passes the signal to downstream componentsMost often - Mitogen Activated Protein Kinase pathways (MAPK pathways)

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MAP kinase regulates the activity of transcription factors Signaling strategies

Receptors that are linked to cytoplasmic enzymesCytokine receptors (tyrosine kinase-linked)

Have the capacity to activate cytosolic tyrosine kinasesReceptor itself lacks kinase activity

Activated kinasephosphorylatescellular substrates

Tyrosine kinase-linked receptors

Have the capacity to activate cytosolic tyrosine kinasesLigand binding causes dimerization of the receptorActivation of cytosolic tyrosine kinase

Receptor itself lacks kinase activityActivated kinase phosphorylates cellular substrates –second messengers

Receptors that activate intracellular tyrosine kinases

Tyrosine kinase-linked receptors

Signal to nucleus through the JAK-STAT pathway (signal transducers and activators of transcription)

Signaling by members of the cytokine receptor family

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Ion channel receptors

Ligand gated ion channelsBinding of a ligand changes the conformation of the receptor and opens channel pore

Ions move through the poreResults in changes of the cell excitability

Termination of signaling

Binding a ligand activates the endocytosis of the receptorsIn endosomes liganddissociates from the receptor based on the pH gradient Receptors got recycled back to the membrane

Cellular mechanisms of steroid hormone action

Steroid hormones

Synthesized from cholesterol in enzymatic reactions in cytosolLipid solubleBind to intracellular receptors

Synthesis of steroid hormones

Will be discussed later when we talk about adrenals

Steroid hormone transport

Lipid soluble hormones require transport proteinsalbumin and transthyretin (prealbumin)specific transport molecules (thyroxine-binding globulin)only unbound hormone can enter the cell !!!

Steroid and thyroid hormones are 99% attached to special transport proteins

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Intracellular receptors

Receptors for hormones that are able to enter the cellLigand activated transcription factorsLocalized in nucleus or cytoplasm

Structure of nuclear receptors

Superfamily of ligand-activated transcription factorsBind to specific DNA sequences as dimersSimilar structure and high homologyTwo highly conserved regions

Domain structure of nuclear receptors

Highest homology region

Mechanism of action of nuclear receptors

In the absence of hormone the DNA binding domain is bound to chaperones (mostly hspfamily)Binding of a hormone causes dissociation of hspfrom a receptor and exposure of DNA binding domain

Hormones that bind to intracellular receptors

All hormones that can cross the membraneSmall hydrophobic molecules

Steroid hormonesThyroid hormones1,25-dihydroxycholecalciferolRetinoic acid

Hormones that bind to intracellular receptors

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Steroid hormone action

http://www.maxanim.com/biochemistry/Steroid%20Hormone/Steroid%20Hormone.swf

Lipid soluble hormones effects

Induce transcription and translationAlter transcription of specific genes Exert mostly long term effects - growth and differentiation, new protein synthesis

Regulation of gene transcription

When not bound to the hormone receptors stay bound to chaperones (mostly hsp family)Binding of a hormone causes dissociation of hsp from a receptor and eexposure of zinc fingersActivated receptors bind to DNAInteract predominantly with specific genomic sequences - hormone responsive elements (HRE)

Localized in the 5’ flanking regions of target genes

Regulation of gene expression by homodimer receptors

Recruitment of a co-activator complexStabilization of preinitiation complex at TATA boxBinding of TFIIB Binding of polymerase

Regulation of gene expression by heterodimer receptors

Regulation of gene expression by cytoplasmic receptors

Glucocorticoid receptors are localized in the cytoplamsIn the absence of hormone cytoplasmic receptor is bound to hsp90Ligand binding displaces hsp90 complex Receptor – ligand complex translocates to the nucleus and binds to Hormone Response Element on DNA

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Regulation of gene expression by glucocorticoid receptor

Eicosanoids

Derivatives of arachidonic acid2 groups – prostaglandins and leukotrienesProstaglandins are produced by COX enzyme (Cox inhibitors are NSID)Important in coordinating tissue responses to injury or diseaseAre important paracrine factors