Mechanisms of Hormone Action

2

General principles:

2. Signals have different chemical natures.

4. Cells respond to sets of signals.

3. The same signal can induce a different response in different cells.

5. Receptors relay signals via intracellular signaling cascades.

1. Signals act over different ranges.

3

• Cells sense and respond to the environment

Prokaryotes: chemicals Humans: light - rods & cones of the eye sound – hair cells of inner ear chemicals in food – nose & tongue

• Cells communicate with each other

Direct contact Chemical signals

External signals are converted to

Internal Responses

General Principles of Signal Transduction Signal transduction refers to the overall process of converting extracellular signals into intracellular responses .

Key players in signal transduction are signaling molecules, receptors, signal transduction proteins and second messengers, and effector proteins.

Cells respond to signals by changing the activity of existing enzymes (fast) and/or the levels of expression of enzymes and cell components (slower) by gene regulation (Steps 7a & 7b).

Receptors and signal transduction systems have evolved to detect and respond to hormones, growth factors,

drugs & neurotransmitters.

6

EXTRACELLULAR

FLUID

Receptor

Signal

molecule

Relay molecules in a signal transduction pathway

Plasma membrane

CYTOPLASM

Activation

of cellular

response

Reception 1 Transduction 2 Response 3

Primary Messenger

Secondary Messengers

Target Enzymes

Cascade Effect

7

Each protein in a signaling pathway – Amplifies the signal by activating multiple

copies of the next component in the pathway

primary signal - activates an enzyme activity, processes 100 substrates /second

Primary enzyme activates 100 target enzymes

Each of the 100 enzymes activates an additional 100 downstream target enzymes

Each of the 10,000 downstream targets

activates 100 control factors so rapidly have

1,000,000 active control factors

8 Glucose-1-phosphate

(108 molecules)

Glycogen

Active glycogen phosphorylase (106) Inactive glycogen phosphorylase

Active phosphorylase kinase (105) Inactive phosphorylase kinase

Inactive protein kinase A

Active protein kinase A (104)

ATP

Cyclic AMP (104)

Active adenylyl cyclase (102)

Inactive adenylyl cyclase

Inactive G protein

Active G protein (102 molecules)

Binding of epinephrine to G-protein-linked receptor

(1 molecule)

Transduction

Response

Reception

1

102

104

105

106

108

A Signal Cascade

amplification

9

Receptors relay signals via intracellular SIGNALING CASCADES

Main Types of Receptors

• ION CHANNEL RECEPTORS

• G-PROTEIN-COUPLED RECEPTORS

• KINASE-LINKED RECEPTORS

• INTRACELLULAR RECEPTORS

11

Trimeric G-protein-linked

Cell-surface receptors -large &/or hydrophilic ligands

ion-channel-linked

enzyme-linked (tyrosine

kinase)

Signal transduction Control of ion channels • Receptor protein is part of an ion channel protein

complex

• Receptor binds a messenger leading to an induced fit

• Ion channel is opened or closed

• Ion channels are specific for specific ions (Na+, Ca2+, Cl-,

K+)

• Ions flow across cell membrane down concentration

gradient

• Polarises or depolarises nerve membranes

• Activates or deactivates enzyme catalysed reactions within cell

13

Ion channel receptors

Cellular

response

Gate open

Gate close

Ligand-gated

ion channel receptor

Plasma

Membrane

Signal

molecule

(ligand)

Gate closed Ions

Examples: Muscle Contraction Nerve Cell communication

14

Review: Remember the Na+/K+ ATPase (Na+/K+ pump)? [Na+] inside ~10mM; outside ~150mM [K+] inside ~100mM; outside ~5mM cell has membrane potential ~ -60mV

-60mV K+ A-

Na+ Cl-

- -

- - -

-

+ + +

+

+ +

15

When a ligand binds to a receptor protein, the

cell has a response.

signal transduction: the events within the

cell that occur in response to a signal

Different cell types can respond differently to

the same signal.

Intercellular Communication

16

A cell’s response to a signal often involves

activating or inactivating proteins.

Phosphorylation is a common way to change

the activity of a protein.

protein kinase – an enzyme that adds a

phosphate to a protein

phosphatase – an enzyme that removes a

phosphate from a protein

Intercellular Communication

17

Signal Transduction Components: Kinases/Phosphatases

Proteins that participate in intracellular signal transduction fall into two main classes--protein kinases/phosphatases and GTPase switch proteins. Kinases use ATP to phosphorylate amino acid side-chains in target proteins. Kinases typically are specific for tyrosine or serine/threonine sites. Phosphatases hydrolyze phosphates off of these residues. Kinases and phosphatases act together to switch the function of a target protein on or off .

Kinases - Phosphorylation

Phosphatase - Dephosphorylation

Tyrosine-OH Tyr-Kinases

Serine-OH Ser/Thr-Kinases

Threonine-OH

„dual specificity“ Kinases

There are about 600 kinases and 100 phosphatases encoded in the human genome. Activation of many cell-surface receptors leads directly or indirectly to changes in kinase or phosphatase activity. Note that some receptors are themselves kinases (e.g., the insulin receptor).

21

Growth hormone receptor

Tetrameric complex constructed in presence of growth

hormone

Growth hormone binding site

Kinase active site

Kinase active site

opened by induced fit

GH

OH OH

OH

HO

kinases

GH receptors

(no kinase activity)

GH binding

&

dimerisation

OP OP OP

PO

ATP ADP

Activation and phosphorylation

OH

Binding of kinases

OH OH

HO

23

Intracellular Receptors

steroid hormones

-have a nonpolar, lipid-soluble structure

-can cross the plasma membrane to a steroid

receptor

-usually affect regulation of gene expression

An inhibitor blocks the receptor from binding to

DNA until the hormone is present.

Intracellular receptors

•Chemical messengers must cross cell membrane

• Chemical messengers must be hydrophobic

• Example-steroids and

steroid receptors

Zinc

Zinc fingers contain Cys residues (SH)

Allow S-Zn interactions

CO2H

H2N

DNA binding region

(‘zinc fingers’)

Steroid

binding region

25

Intracellular Receptors

A steroid receptor has 3 functional domains:

1. hormone-binding domain

2. DNA binding domain

3. domain that interacts with coactivators to

affect gene expression

26

Structure of GPCRs G protein-coupled receptors (GPCRs) are the most numerous class of receptors in most eukaryotes. Receptor activation by ligand binding activates an associated trimeric G protein, which in turn interacts with downstream signal transduction proteins. All GPCRs are integral membrane proteins that have a common 7 transmembrane segment structure . The hormone/ligand binding domain is formed by amino acids located on the external side of the membrane and/or membrane interior . GPCRs interact with G proteins via amino acids in the C3 and C4 cytoplasmic regions.

Biological functions mediated by 7TM receptors Smell Taste Vision Neurotransmission Hormone secretion Exocytosis Control of blood pressure Embryogenesis Cell growth and differentiation Development Viral infection Carcinogenesis

G Protein Activation of Effectors

The trimeric G protein cycle of activity in hormone-stimulated GPCR regulation of effector proteins is summarized in (next slide).

Trimeric G Proteins & Their Effectors There are 21 different Ga proteins encoded in the human genome. The G proteins containing these subunits are activated by different GPCRs and regulate a variety of different effector proteins . The most common effectors synthesize second messengers such as cAMP, IP3, DAG, and cGMP. In the case of cAMP, a stimulatory Gas subunit activates adenylyl cyclase and cAMP production, whereas an inhibitory Gai subunit inhibits adenylyl cyclase and cAMP production.

GPCRs that Regulate Adenylyl Cyclase Adenylyl cyclase is an effector enzyme that synthesizes cAMP. Ga-GTP subunits bind to the catalytic domains of the cyclase, regulating their activity. Gas-GTP activates the catalytic domains, whereas Gai-GTP inhibits them. A given cell type can express multiple types of GPCRs that all couple to adenylyl cyclase. The net activity of adenylyl cyclase thus depends on the combined level of G protein signaling via the multiple GPCRs. In liver, GPCRs for epinephrine and glucagon both activate the cyclase. In adipose tissue , epinephrine, glucagon, and ACTH activate the cyclase via Gas-GTP, while PGE1 and adenosine inactivate the cyclase via Gai-GTP.

Activation of Gene Transcription by GPCR Signaling

GPCRs regulate gene transcription by cAMP and PKA signaling. cAMP-released PKA catalytic domains enter the nucleus and phosphorylate the CREB (CRE-binding) protein, which binds to CRE (cAMP-response element) sequences upstream of cAMP-regulated genes. Only phosphorylated p-CREB has DNA binding activity. p-CREB interacts with other TFs to help assemble the RNA Pol II transcription machinery at these promoters. In liver, glucagon signaling via this pathway activates transcription of genes needed for gluconeogenesis.

G proteins are important signal transducing molecules in

cells.

"Malfunction of GPCR [G Protein-Coupled Receptor]

signaling pathways are involved in many diseases, such

as diabetes, blindness, allergies, depression, cardiovascular

defects, and certain forms of cancer.

It is estimated that about 30% of the modern drugs' cellular

targets are GPCRs." The human genome encodes roughly

800 G protein-coupled receptors, which detect photons of

light, hormones, growth factors, drugs, and other

endogenous ligands. Approximately 150 of the GPCRs found

in the human genome have still-unknown functions.

Whereas G proteins are activated by G protein-coupled

receptors, they are inactivated by RGS proteins (for "Regulator of G

protein signalling"). Receptors stimulate GTP binding (turning the G

protein on). RGS proteins stimulate GTP hydrolysis (creating GDP, thus

turning the G protein off).

A number of events contribute to the termination of signaling by a GPCR. These include:

1. dissociation of the hormone from the receptor,

2. hydrolysis of GTP by Ga

3. hydrolysis of cAMP via cAMP phosphodiesterase,

4. phosphorylation and “desensitization” of receptors by kinases such as PKA and ß-adrenergic receptor kinase (BARK).

5. In addition, GPCRs can be removed from the membrane by vesicular uptake.

Down-regulation of GPCR/cAMP/PKA Signaling

39

G-protein activation

“molecular switch”

(b) Ligand binds G-protein associates (c) GDP-GTP exchange •a-Subunit dissociates

inactive

Active G-Protein-GTP -> allosteric modulator of target effector enzyme

active

40

•There are TWO broad subclasses of trimeric G-protein-activated signal transduction pathways: depends on their target effector enzymes A. adenylyl cyclase B. phospholipase C

All G-proteins – similar structure/activation

41

An activated Ga-protein-GTP – Can trigger the formation of cAMP, which then acts as a second messenger in cellular pathways

ATP

GTP

cAMP

Protein

kinase A

Cellular responses

G-protein-linked

receptor

Adenylyl

cyclase G protein

First messenger

(signal molecule

such as epinephrine)

42

G-protein-GTP activation of

Effector Enzyme adenylyl cyclase produces the 2nd messenger cAMP

Activated G-protein

Adenylyl Cyclase & Protein Kinase A Adenylyl cyclase is an integral membrane protein that contains 12 transmembrane segments . It also has 2 cytoplasmic domains that together form the catalytic site for synthesis of cAMP from ATP. One of the primary targets of cAMP is a regulatory kinase called protein kinase A (PKA), or cAMP-dependent protein kinase.

PKA exists in two different states inside cells . In the absence of cAMP, the enzyme forms a inactive tetrameric complex in which 2 PKA catalytic subunits are non-covalently associated with 2 regulatory subunits. When cAMP concentration rises, cAMP binds to the regulatory subunits which undergo a conformational change, releasing the active catalytic subunits.

45

inactive

+ P active

Phosphorylase kinase

Protein Kinase A Phosphorylates downstream target enzymes

Breaks down Glycogen

Into Glucose

46

cAMP regulated pathways

Function target tissue signal Glycogen breakdown muscle epinephrine Glycogen breakdown liver glucagon

Heart rate cardiovascular epinephrine Water reabsorption kidney antidiuretic hormone

What are targets for Protein Kinase A??

47

How to shut it off?

Auto Shut-off

G-protein a-subunit is on a timer

Inherent GTPase activity

No ligand

48

How to shut it off?

cAMP-phosphodiesterase rapidly cleaves

cAMP (so short lived)

49

What if you can’t turn off cascade?

Vibrio cholera - causes cholera

Normal gut: H20, NaCl, NaHCO3 secretion controlled by hormones via Gs/cAMP signal pathways

V. cholera – secretes enterotoxin, chemically modifies aGs – no GTPase activity - stays ON Severe watery diarrhea – dehydration, death

50

target effector enzyme is Phospholipase C

PLC cleaves a membrane phospholipid (Phoshatidyl inositol) to

two 2nd Messengers:

Inositol-1,4,5-Trisphosphate (InsP3) &

Diacylglycerol (DAG)

51

InsP3 Water Soluble

DAG Lipid

Soluble

PIP2

GPCRs That Activate Phospholipase C Another common GPCR signaling pathway involves the activation of phospholipase C (PLC). This enzyme cleaves the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2) to the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Fig.). In this case, the Gao(other) and Gaq Ga proteins conduct the signal from the GPCR to PLC. This is the pathway used in

a1-adrenergic GPCR signaling in the liver.

*

IP3/DAG Signaling Elevates Cytosolic Ca2+ The steps downstream of PLC that make up the IP3/DAG signaling pathway are illustrated in the figure. IP3 diffuses from the cytoplasmic membrane to the ER where it binds to and triggers the opening of IP3-gated Ca2+ channels (Steps 3 & 4). Another kinase, protein kinase C (PKC) binds to DAG in the cytoplasmic membrane and is activated (Step 6). In liver, the rise in cytoplasmic [Ca2+] activates enzymes such as glycogen phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase. Glycogen phosphorylase kinase is activated by Ca2+-calmodulin. In addition, PKC phosphorylates and inactivates glycogen synthase.

55

DAG Activates

Protein Kinase C

(Starts Cascade)

InsP3 Ligand for ER

ligand- gated Ca++

channels Ca++ levels

56

Protein Kinase C phosphorylates target proteins (ser & thr) cell growth regulation of ion channels cytoskeleton increases cell pH Protein secretion

Ca++ Binds & activates calmodulin Calmodulin-binding proteins activated (kinases & phosphatases)

Response:

Signal Trans. Components: GTPase Switches

GTPase switch protein also play important roles in intracellular signal transduction . GTPases are active when bound to GTP and inactive when bound to GDP. The timeframe of activation depends on the GTPase activity (the timer function) of these proteins. Proteins known as guanine nucleotide-exchange factors (GEFs) promote exchange of GTP for GDP and activate GTPases. Proteins known as GTPase-activating proteins (GAPs), stimulate the rate of GTP hydrolysis to GDP and inactivate GTPases.

.

We will cover two classes of GTPase switch proteins--trimeric (large) G proteins, and monomeric (small) G proteins. Trimeric G proteins interact directly with receptors, whereas small G proteins interact with receptors via adaptor proteins and GEFs.

Signal Trans. Components: 2nd Messengers While there are a large number of extracellular receptor ligands ("first messengers"), there are relatively few small molecules used in intracellular signal transduction ("second messengers"). In fact, only 6 second messengers occur in animal cells. These are cAMP, cGMP, 1,2-diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP3) , and calcium and phosphoinositides . Second messengers are small molecules that diffuse rapidly through the cytoplasm to their protein targets. Another advantage of second messengers is that they facilitate amplification of an extracellular signal.

Nitric Oxide (NO)/cGMP Signaling A related signaling pathway involving phospholipase C operates in vascular endothelial cells and causes adjacent smooth muscle cells to relax in response to circulating acetylcholine (Fig.) In the NO/cGMP signaling pathway, the downstream target of Ca2+/calmodulin is nitric oxide synthase, which synthesizes the gas NO from arginine. NO diffuses into smooth muscle cells and causes relaxation by activating guanylyl cyclase and increasing [cGMP]. As a result arteries in tissues such as the heart dilate, increasing blood supply to the tissue. NO also is produced from the drug nitroglycerin which is given to heart attack patients and patients being treated for angina.

10-7 M

10-3 M

“Second messenger“

DAG, IP3 and Ca++

Metabolism of Diacylglycerol. Diacylglycerol may be (1) phosphorylated to phosphatidate or (2) hydrolyzed to glycerol and fatty acids.

65

Summary - signaling is endocrine, paracrine, synaptic, or direct cell contact - signal transduction is mediated by receptor proteins - Receptors bind primary signal (ligand) - Some amplification event occurs - Example: ligand gated ion channel opens influx of ions triggers change in activity (vesicle fusion in nerve end, contraction in muscle) - Example: ligand binds to 7-pass membrane receptor

catalyzes GTP exchange to Ga-subunit of trimeric G-protein active Ga-subunit-GTP is allosteric activator of effector enzymes: - ADENYLATE CYCLASE: makes cyclic AMP - PHOSPHOLIPASE C: makes DAG and IP3 these second messengers activate target enzymes Trigger cascades

- Must shut off cascade: removal of ligand, hydrolysis of GTP, phosphodiesterase, protein phosphatases, Ca++ ion pumps