Mechanisms of hormone action

Hormones 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 substance High affinity and specificity of binding to specific receptors on target cells Initiates biological response

Function of hormones

Life of a hormone

HOMEOSTASIS Reproduction Growth and development Maintenance of internal environment Production, utilization and storage of energy
Hormone transport protein

Chemical nature of hormones

Amino acid derivatives

Derivatives of tyrosine Catecholamines (epinephrine, dopamine) Thyroid hormones (dipeptides) Tryptophan derivative Melatonin

Can be divided into 3 groups Amino acid derivatives Peptide hormones Lipid derivatives

Peptide hormones

2 classes of lipid derived hormones

Glycoproteins from anterior pituitary thyroid-stimulating hormone (TSH) luteinizing hormone (LH) follicle-stimulating hormone (FSH) Peptides and small proteins Digestive tract hormones Pituitary hormones Pancreatic hormones

Steroid hormones: derived from cholesterol 2 groups with the intact steroid ring (adrenal and gonadal steroids) with the steroid ring cleaved (metabolites of vit D) Eicosanoids: derived from arachidonic acid

Catecholamines

Synthesis of catecholamines

Molecules with catechol group Hormonal regulators Dopamine in hypothalamus inhibits prolactin secretion Epinephrine (adrenaline) stress reaction Synthesized from aa phenylalanine or tyrosine in enzymatic reactions

Synthesized in cytosol Packaged in vesicles and exocytosed Water soluble, do not need transport proteins Work from the outside from the cell (bind to surface receptors)

Synthesis of melatonin (indoleamine)

Peptide hormone synthesis

Tryptophan to serotonin NAT (N-acetyltransferase) is activated only in the dark Works on surface receptors

Synthesized on the ribosomes attached to rough ER All of them have ER targeting sequence on the Nterminus (signal peptide) Synthesized as prohormones Inactive molecules converted to active hormones in ER and Golgi, sometimes after secretion Exocytosed from the cell Work from the outside from the cell (bind to surface receptors)

Cell biology vs. endocrinology

Splice variants Alternative processing of mRNA can result in splice variants of the same hormone Availability of transcription factors can affect hormone production

Preprohormone full sequence of the peptide Prohormone peptide minus signal sequence Can (and usually does) undergoes additional proteolytic cleavage in Golgi Hormone convertase Hormone biologically active product

Posttranslational processing

Posttranslational processing

Signal peptide removal Folding (ER) Formation of disulfide bonds Glycosylation (ER)

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

Peptide homology

Neurohypophyseal hormones

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

Shape matters

Peptide hormone transport

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

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

Signaling process

Hormone receptors

Recognition of signal Receptors Transduction Change of external signal into intracellular message Effect Modification of cell behavior

Molecules within or on the surface of target cells that bind hormones with high affinity and specificity and thereby initiate and mediate biological responses Hormones will only produce the response in cells that express the receptors for this particular hormone (target cells) ONLY target cells respond to hormone Cells that do not have receptors for the hormone ignore the hormone

Properties of the hormone-receptor interactions

Peptide and amine receptors

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

Surface receptors a.k.a transmembrane receptors Peptides 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

Many families of cell surface receptors

G protein coupled receptors

Based on homology and signaling strategy The same ligand can bind to two or more different families!!! Multiple splice variants (1 and 2 adrenergic receptors) can be tissue specific

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

G protein coupled receptors = GPCR

Molecular properties of G protein coupled receptors

Ligand binding to the receptor activates a signal transduction cascade that comprises G protein molecular switch Enzyme that produces second messengers Second messengers Target protein - effector But not necessary all steps are involved!!!!

A.k.a. serpentine receptors Seven transmembrane regions of 22-24 hydrophobic residues N-terminus faces outside (ligand binding domain) C-terminus faces cytosol A cytosolic loop between helices 5 and 6 is the place for interaction with G protein

G proteins

G protein cycle

Membrane bound heterotrimeric proteins consisting of 3 subunits , , Coupled to surface receptors Molecular switches Use the exchange and hydrolysis of nucleotides (GTP/GDP) to transduce the signal from the surface receptors to intracellular effectors

When G protein is inactive it is bound to GDP and exists as a trimer The exchange of GDP for GTP activates G protein G protein dissociates into two subunits: and dimer GTP is bound to subunit Subunit has an intrinsic GTPase activity and hydrolyses GTP to GDP This process terminates the signal and reassociate

What the G proteins can do?

Amplification of signal by second messengers

Activate enzymes to produce second messengers Activate transcription factors Modulate ion channels, pumps and exchangers Affect cytoskeleton Modulate enzymes

Adrenaline signaling

Differential regulation of adenylate cyclase

Regulation of transcription by cAMP kinase

CREB needs to be phosphorylated at serine 133

Activated by Gs Inhibited by Gi

Only genes that have CRE sequence are activated by those receptors

Activated CREB binds to CRE sequence and stimulates transcription

CREB links cAMP signals to transcription

Second messengers

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

cAMP is not the only second messenger initiated by GPCRs IP3 (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 IP3 and DAG Several other second messenger are derived from membrane lipids

PIP2 breakdown

IP3 as a second messenger

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

Calcium is also intracellular second messenger

Protein kinase C (PKC) signaling

Regulation of hormone secretion Regulation of transcription through Ca- calmodulin kinase

Serine/threonine kinase Activated by DAG Phosphorylates various cellular effectors Activates transcription factors AP-1 (c-fos and c-jun are both protooncogenes)

Other lipid messengers

Ceramide signaling
Product of sphingomyelin cycle Sphingomyelins do not have glycerol backbone Second messenger in TNF- signaling and stimulation of apoptosis Increase in prostaglandin biosynthesis Activation of transcription factor NF b

Catalytic receptors with intrinsic enzymatic activity

Receptor tyrosine kinases

Ligand binding causes activation of enzymatic activity of the receptor (receptor itself is an enzyme) Tyrosine kinase Guanylyl cyclase Phosphatase Modification of cellular activity

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

RTKs

Signaling by RTK

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

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

Enzymes activated by RTK

How do RTKs activate MAPK pathways and affect transcription?

Phosphotyrosine residues on the kinase interact with adapter proteins Transmit a signal to Ras, a monomeric G protein (molecular switch) Ras passes the signal to downstream components Most often - Mitogen Activated Protein Kinase pathways (MAPK pathways)

MAP kinase regulates the activity of transcription factors

Signaling strategies
Receptors that are linked to cytoplasmic enzymes Cytokine receptors (tyrosine kinase-linked) Have the capacity to activate cytosolic tyrosine kinases Receptor itself lacks kinase activity Activated kinase phosphorylates cellular substrates

Tyrosine kinase-linked receptors

Receptors that activate intracellular tyrosine kinases

Have the capacity to activate cytosolic tyrosine kinases Ligand binding causes dimerization of the receptor Activation of cytosolic tyrosine kinase Receptor itself lacks kinase activity Activated kinase phosphorylates cellular substrates second messengers

Tyrosine kinase-linked receptors

Signaling by members of the cytokine receptor family

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

Ion channel receptors

Termination of signaling

Ligand gated ion channels Binding of a ligand changes the conformation of the receptor and opens channel pore Ions move through the pore Results in changes of the cell excitability

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

Steroid hormones

Cellular mechanisms of steroid hormone action

Synthesized from cholesterol in enzymatic reactions in cytosol Lipid soluble Bind to intracellular receptors

Synthesis of steroid hormones

Steroid hormone transport

Will be discussed later when we talk about adrenals

Lipid soluble hormones require transport proteins albumin 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

Structure of nuclear receptors

Receptors for hormones that are able to enter the cell Ligand activated transcription factors Localized in nucleus or cytoplasm

Superfamily of ligand-activated transcription factors Bind to specific DNA sequences as dimers Similar structure and high homology Two highly conserved regions

Domain structure of nuclear receptors

Mechanism of action of nuclear receptors

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

Highest homology region

Hormones that bind to intracellular receptors

Hormones that bind to intracellular receptors

All hormones that can cross the membrane Small hydrophobic molecules Steroid hormones Thyroid hormones 1,25-dihydroxycholecalciferol Retinoic acid

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Steroid hormone action

Lipid soluble hormones effects

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

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

Regulation of gene transcription

Regulation of gene expression by homodimer receptors

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 fingers Activated receptors bind to DNA Interact predominantly with specific genomic sequences - hormone responsive elements (HRE) Localized in the 5 flanking regions of target genes

Recruitment of a co-activator complex Stabilization of preinitiation complex at TATA box Binding 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 cytoplams In the absence of hormone cytoplasmic receptor is bound to hsp90 Ligand 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 acid 2 groups prostaglandins and leukotrienes Prostaglandins are produced by COX enzyme (Cox inhibitors are NSID) Important in coordinating tissue responses to injury or disease Are important paracrine factors

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