SECOND MESSENGERS

Presenter : Dr.Anu Priya J

SCHEME

• History• Introduction• Types• cAMP Pathway• cGMP Pathway

• IP3 / DAG Pathway

• Calcium as a second messenger• Eicosanoids• Applied aspects

HISTORY

• Earl Wilbur Sutherland Jr. – 1971 Nobel Prize in Physiology or Medicine

• Epinephrine – liver - glycogen to glucose – cAMP

• Martin Rodbell & Alfred G. Gilman – 1994 Nobel Prize

HISTORY

INTRODUCTION

• Cell to cell communication

• Chemical and physical messengers interact with receptors in the plasma membrane , cytoplasm or nucleus.

• A series of signaling events that mediate the response to each stimulus.

• Response – specific, amplified ,tightly regulated and coordinated.

Plasma membrane

EXTRACELLULARFLUID

CYTOPLASM

Reception Transduction Response

Receptor

Signalingmolecule

Activationof cellularresponse

Relay molecules in a signal transductionpathway

321

Earl Sutherland – cell signaling- 3 processes

• Signaling pathways are characterized by:

1. Multiple, hierarchical steps

2. Amplification of the hormone-receptor binding event which magnifies the response

3. Activation of multiple pathways and regulation of multiple cellular functions

4. Feedback mechanisms – tight regulatory control

TYPES

• Hydrophobic molecules

-Membrane associated

-Phosphatidylinositol , Diacylglycerol

• Hydrophilic molecules

-Cytosolic

- cAMP, cGMP, inositol triphosphate,Ca2+

• cAMP pathway

• cGMP pathway

• IP3 / DAG pathway

• Calcium as a second messenger

• Eicosanoids

Second Messengers

11

Second Messengers

• General characteristics– Low amounts in resting state– Regulated synthesis– Regulated destruction– Act through other proteins

Hormone receptor

GDP

G protein

GTP

Adenylcyclase

cAMP

ATP

cAMP pathway

cAMP pathway

cAMP-dependent Protein Kinase A

C

R R

C

+ 4 cAMPC

C R

R

cAMP

cAMP

cAMP

cAMP

+ The catalytic subunit is now free to attack a protein target.

cAMP pathway

Active phosphorylaseKinase

Inactive PhosphorylaseKinase

P

P

Catalytic site

Calmodulin

2ATP 2ADP

cAPK

Kinase enzymes are the targets for the catalytic subunit of PKA

These target enzymes have some unique features.

Phosphorylase kinase, for example, is composed of 4 different subunits . The delta subunit is calmodulin, a calcium binding protein, that regulates the activity. The gamma subunit has the catalytic site . The enzyme is not active.

To activate phosphorylase kinase, the catalytic subunit of protein kinase A transfers 2 PO4s from 2 ATPs to the alpha and beta subunits. Phosphorylation at these sites renders phosphorylase kinase active. Conversely, removing phosphate inactivates the kinase.

P

The target kinases phosphorylate enzymes that control critical steps in a pathway. For example, phosphorylase or glycogen synthase are targets of protein kinase.

For phosphorylase, the kinase enzyme is phosphorylase kinase.

The sequence of events is highlighted in the figure below

Adenylcyclase

C C

R R

cAMP dependentprotein kinase

Phosphorylasekinase Phosphorylase

Thus hormones(external to the cell) have profound effect on glycogen degradation, glycogen synthesis, and other processes by controlling the enzyme activities by the presence or absence of phosphate groups.

P

cAMP pathway

cAMP pathway

• In addition to signaling in the cytoplasm, the catalytic subunit of PKA can enter the nucleus of cells and phosphorylate and activate the transcription factor cAMP response element binding (CREB) protein.

• Phospho-CREB protein increases the transcription of many genes

• Indirect effect of cAMP mediated by PKA

cAMP pathway

cyclic AMP mediated response

Epinephrine (α2,β1,β2)

Acetylcholine(M2)

NorepinephrineGlucagonSomatostatin ACTHCRHADH

FSHhCGLHMSH  PTHTSHAngiotensin IICalcitonin

cGMP Pathway

Ligand-receptor

Guanyl cyclase

GTP cGMP

Protein kinase G Active protein kinase G

Effects

PDE

GMP

22

• smooth muscle relaxation• vision• ANP,NO

cGMP Pathway

• cGMP Pathway in phototransduction

cGMP Pathway

Nitric oxide (NO)

NO, a simple gas, is able to diffuse across the membrane, and alters the activity of intracellular target enzymes. It’s extremely unstable, so its effects are local. Ex. It signals the dilation of blood vessels.

Mechanism. Acetylcholine is released from the terminus of nerve cell in the blood vessel wall. The endothelial cells are stimulated to produce NO (from arginine), which causes an increased synthesis of cGMP, a second messenger responsible for blood vessel dilation.

Ach

Nerve cell endothelial cell

NO cGMP Vessel dilationAchR

cGMP Pathway

IP3 /DAG

LIGAND RECEPTOR

↓

G protein

↓

Phospholipase C

↓

PIP2 IP3 + DAG

↓

Endoplasmic reticulum opening of Ca channels

↓

Ca++

↓

Protein kinase C Effects

G protein

EXTRA-CELLULARFLUID

Signaling molecule(first messenger)

G protein-coupledreceptor

Phospholipase C

DAG

PIP2

IP3

(second messenger)

IP3-gatedcalcium channel

Endoplasmicreticulum (ER)

CYTOSOL

Ca2

GTP

Figure 11.14-1

Figure 11.14-2

G protein

EXTRA-CELLULARFLUID

Signaling molecule(first messenger)

G protein-coupledreceptor

Phospholipase C

DAG

PIP2

IP3

(second messenger)

IP3-gatedcalcium channel

Endoplasmicreticulum (ER)

CYTOSOL

Ca2

(secondmessenger)

Ca2

GTP

Figure 11.14-3

G protein

EXTRA-CELLULARFLUID

Signaling molecule(first messenger)

G protein-coupledreceptor

Phospholipase C

DAG

PIP2

IP3

(second messenger)

IP3-gatedcalcium channel

Endoplasmicreticulum (ER)

CYTOSOL

Variousproteinsactivated

Cellularresponses

Ca2

(secondmessenger)

Ca2

GTP

IP3 /DAG

30

• Inositol tri-phosphate• Hydrophilic• Agonist for internal calcium channel

• [Ca++]i rises

• Multiple effects through Ca++-binding proteins

• Diacylglycerol• Hydrophobic • Targets PKC (a

kinase)• PKC requires Ca++

and DAG

IP3 /DAG

• Classical PKC family members (PKCα,PKCβ,PKCγ) require both Ca2+ & DAG for activation.

• The novel PKCs (PKCδ,PKCε,PKCη) are independent of Ca2+

IP3 /DAG

Epinephrine (α1) Acetylcholine (M1,M3) Angiotensin GnRH  GHRH  Oxytocin  TRH  PDGF

IP3 /DAG

• Calcium ions - once they enter the cytoplasm exert allosteric regulatory effects on many enzymes and proteins.

• Calcium acts as a second messenger by indirect signal transduction pathways such as via G protein-coupled receptors.

Calcium as a 2nd Messenger

• Low cytoplasmic Ca++ at rest (10–100 nM).

• To maintain this low concentration, Ca2+ is actively pumped from the cytosol to the extracellular space and into the endoplasmic reticulum (ER)

• Certain proteins of the cytoplasm and organelles act as buffers by binding Ca2+.

• Signaling occurs when the cell is stimulated to release calcium ions (Ca2+) from intracellular stores, and/or when calcium enters the cell through plasma membrane ion channels.

Calcium as a 2nd Messenger

• sudden increase in the cytoplasmic Ca2+ level up to 500–1,000 nM by opening channels in the endoplasmic reticulum or the plasma membrane.

• Phospholipase C  pathway – IP3 & DAG

• Eicosanoids

Calcium as a 2nd Messenger

• Many of Ca2+-mediated events occur when the released Ca2+ binds to and activates the regulatory protein calmodulin.

• Calmodulin may activate calcium-calmodulin-

dependent protein kinases, or may act directly on other effector proteins.

• Besides calmodulin, there are many other Ca2+-binding proteins such as troponin C that mediate the biological effects of Ca2+.

Calcium as a 2nd Messenger

Calcium as a 2nd Messenger

38

Calmodulin Targets

• Adenylate cyclase

• Phosphodiesterase

• Myosin light chain kinase

• Calmodulin-dependent kinases

• Calcineurin (a phosphatase)

This class of lipids act as signaling molecules that bind to cell surface molecules.

They include: PROSTAGLANDINS PROSTACYCLIN TROMBOXANES LEUKOTRIENES.

The eicosanoids are rapidly broken down and therefore act in autocrine or paracrine pathways. They stimulate a variety of responses in their target cells, including blood platelet aggregation, inflammation, and smooth musclecontraction.

EICOSANOIDS

EICOSANOIDS

Direct pathway

Serotonin(5HT2)

Glutamate(mGLUR1)

Fibroblast growth factor β IFN α IFN γ

Indirect pathway

Dopamine(D2)

Adenosine(A1)

Norepinephrine(α2)

Serotonin(5HT1)

EICOSANOIDS

• Caffeine and methylxanthines inhibit cAMP phosphodiesterases

• Thus prolong cellular response mediated by cAMP and PKA

Applied

• Cholera toxin – ADP ribosylation- Gαs

• Pertussis toxin – ADP ribosylation- Gαi

Applied

• McCune-Albright syndrome

- A somatic mutation that constitutively activates the Gαs in a mosaic pattern

- Excess cAMP- Characteristic triad

i. Variable hyperfunction of multiple endocrine glands, including precocious puberty in girls

ii. Bone lesions

iii. Pigmented skin lesions (café au lait spots)

Applied

Huntington’s disease

• Disturbance of CREB protein in the brain can contribute to the development and progression of Huntington’s disease.

• Autopsied brains of those who had Huntington's disease have been found to have incredibly reduced amounts of CREB protein

Applied

Applied

Angina pectoris

Nitroglycerine

↓

Nitric oxide

↓

cGMP

↓

Protein kinase G

↓

Relax smooth muscle in coronary arteries

Huntington’s disease

• Mutant Htt→ IP3 receptors more sensitive to IP3 → increased release of Ca2+ from ER→increase in cytosolic and mitochondrial concentration of calcium→ Ca2+ induced degeneration of GABAergic medium spiny neurons

Alzheimer's Disease• Familial – mutation of PS1,PS2,APP genes →increased IP3

mediated calcium release

Applied

Eicosanoids are synthesized from arachidonic acid. The first enzyme involved in their synthesis (cyclooxygenase, COX) is the target of ASPIRIN.

Aspirin actions:-reduces inflammation and pain (inhibition of prostaglandins)- reduces platelet aggregation and blood clotting (thromboxanes)

Applications:- prevention of stroke

AA COX aspirin

P

T

Applied

THANK YOU

References

• Berne & Levy - Physiology, 6th Edition

• Boron & Boulpaep - Medical Physiology, 2nd Edition

• William’s textbook of Endocrinology,10th edition

• Internet references