Signaling

Biochemistry Free For All

Signaling
• Outline

Background
Membranes
Hormones & Receptors
Second Messengers
Signaling
• Introduction

Needs of a Multicellular Organism

Responding to Environment
Food Status
Danger
Growth
Injury
Signaling
• Introduction

Coordination of Cellular/Tissue Responses

Signaling
Signaling
• Membranes

Lipid Bilayer Prevents Entry of Most Molecules

Lipid Bilayer

Organization of Bilayer
Around Cell

Information must move across membrane

Signal Transduction
Signaling Simplest Signaling

Signal Response
Nerve System Signaling
Hormones and Signal Transduction
• Hormones

Hormones Communicate Messages

Hormones are Made in One Part of the Body
Exert Effects in Other Part of Body
First Messengers of a Multi-component Message
Numerous Chemical Forms

Thyroid Hormone

Epinephrine

Epidermal Growth Factor

Progesterone
Hormones and Signal Transduction
• Introduction

Cellular Signaling Has Complexity

Responses Aimed at Benefitting Organism
 Receptor Binding Outside of Cell

Non-steroid Hormone Numerous Internal

Hormones and Signal Transduction Message Carriers
• Binding to Receptor

Steroid Hormones Diffuse Through Membrane

Cytosolic Receptor

Receptor Binding
Inside Cell
Hormones and Signal Transduction
• Receptors

Interaction of Hormone with Receptor Changes Receptor

Receptor Change Alters Interactions with Other Proteins

Changes on Hormone Binding

Hormones and Signal Transduction
• Membrane-bound Receptors

G-Protein Coupled Receptor

Hormones and Signal Transduction
G-protein Coupled Receptors (GPCRs)

Almost 800 Genes in Human Genome

460 Olfactory Extracellular
β-adrenergic Receptor Domain

Intracellular
Domain

Seven Transmembrane
Membrane Bound Domains
 Binding of Hormone
Hormone
Structural Change of Receptor
GPCR Action Cycle

Return to
Original
State

Replacement of GDP by GTP

Activation of an Enzyme
 Hormones and Signal Transduction
• Receipt of Message

Membrane Receptor Proteins Internalize Message

Activate Synthesis of Second Messengers
Covalent Modification of “Downstream”
Proteins
Alteration of Gene Expression
Change of Enzyme Activities
Inositol 1,4,5 Trisphosphate (IP3)

Ca++ Ca++
Ca++ Ca++
Ca ++
Ca++

Cyclic AMP (cAMP) Calcium Ions

Cyclic GMP (cGMP)
Hormones and Signal Transduction
• GPCRs and G-Proteins

G-Proteins Bind Guanine Nucleotides (GDP and GTP)

Heterotrimeric - α,β,γ Subunits
Associate with GPCRs
Altered by GPCR’s Binding of Hormone

β
Epi

Ep i
γ
n ep

n ep
Epinephrine
hrin

hrin
e

e
GPCR
β
GPCR GPCR +
γ
Resting State GTP GDP Active
Hormones and Signal Transduction
β-adrenergic Receptor Signaling

Creation of the Second Messenger

Second
ATP Messenger
Adenylate
Adenylate
Cyclase
Cyclase

cAMP + PPi

Also Membrane Bound

Activated by binding to
α-subunit of G-protein

Transmits Signal
 Hormones and Signal Transduction
• Actions of the Second Messenger
Catalytic Subunits
Regulatory Subunits

Activated
Protein 4 cAMP
Kinase A
Activity
Altered
by
Phosphorylation
(Inactive)
Hormones and Signal Transduction
• G-Protein Coupled Receptors Outline

Receptor
G-Proteins
Protein Kinase A
Kinase Cascade
Turning Signal Off
The Coffee Connection
Hormones and Signal Transduction
• β-adrenergic Receptor Signaling

3 cAMP
2 ATP
4
PKA PKA-Reg Protein Kinase A
5a
5
PK-P PK Phosphorylase Kinase
Glycogen
Synthase
GS-P GS
6
P-GP-a GP-b Glycogen Phosphorylase
7
Glycogenx-1 Glycogenx

Glucose-1-phosphate
Hormones and Signal Transduction
• PKA Activation
Hormones and Signal Transduction
• Turning Off β-adrenergic Receptor Signaling

Turning off the Signaling Pathway

β-adrenergic Receptor
G-Protein
cAMP
Protein Kinase A
Hormones and Signal Transduction
• Turning Off β-adrenergic Receptor Signaling

Turning Off β-adrenergic Receptor

Exterior of Cell

ATP ADP

G-Protein
Receptor Arrestin

PO
PO
Kinase

4 =
4 =

Cytoplasm

Blocks and
Favors Endocytosis
Hormones and Signal Transduction
• β-adrenergic Receptor Signaling

G-protein Inactivation

Auto-regulating
Inherent GTPase Activity

β
γ
Hydrolysis
β
γ
Phosphate

Return to Resting State

Adenylate Cyclase Inactivated - no more cAMP
Hormones and Signal Transduction
• β-adrenergic Receptor Signaling

PO4=

PO4=

X
Hormones and Signal Transduction
• β-adrenergic Receptor Signaling

PO4=
cAMP Ph
o sp
ho
die
ste
ras
PKA e

AMP
PK-P
GS-P

P-GP-a Becomes Inactive

Glycogenx-1 Glycogenx

Glucose-1-phosphate
Hormones and Signal Transduction
• β-adrenergic Receptor Signaling

PO4=
cAMP Ph
o sp Phosphodiesterase is
ho
die
ste Inhibited by Caffeine
ras
PKA e

AMP
PK-P
GS-P

P-GP-a Drinking Coffee Gives

a Small Boost to
Glycogenx-1 Glycogenx Blood Glucose by
Keeping cAMP Levels
Higher
Glucose-1-phosphate
Receptor Tyrosine Kinases (RTKs)

RTKs are Membrane Bound Proteins that Phosphorylate Tyrosines

RTKs Play Important Roles in Regulating Cell Proliferation
Dimerization Important for Activity

ATP ADP

RTK
Receptor Tyrosine Kinases (RTKs)

Outside of Cell

RTK Monomer RTK Monomer

Lipid Bilayer

Transmembrane α-helix Cytoplasmic Tyrosine

Inside of Cell Kinase Domain
(Cytoplasm) (inactive)
Receptor Tyrosine Kinases (RTKs)

Ligand
(Hormone)
Binding &
Dimerization

Activation of Tails
Autophosphorylation P P
P P
Active Tyrosine
PP
Kinase
Receptor Tyrosine Kinases (RTKs)

Assembly of
Signaling Complex

P P P P
P P P P
PP PP

SH2 Domains of
Signaling Complex
Proteins Recognize and
Communicates Message to
Bind Phosphotyrosines
Cell (usually by phosphorylation)
Receptor Tyrosine Kinases (RTKs)
• RTK Signaling Overview

Binding of Hormone to
RTK in Membrane
Receptor
Dimerization

Autophosphorylation

Signaling
Complex
Assembly

Communicate
Message
to Cell
 RTKs - Insulin Receptor

Unlike Other RTKs, Always a Dimer in Membrane

Binding of Insulin Activates Autophosphorylation of Tails

Other
Signaling Pathways

Binding of Autophos- IRS-1 PI3 Kinase PIP3 PDK1 Akt Kinase

Insulin phorylation Activation Activation Formation Activation Activation

Insulin Signaling Also Blood Stimulate

Activates Cells GLUT4
Glucose Uptake
Phosphoprotein Levels Movement to
Phosphatase Glucose Cytoplasm
Fall
 Glycogen Made
PK, GP-a Inactive GS Active

Hormones and Signal Transduction Phosphoprotein Phosphatase

Activated

β- Adrenergic Pathway Blood Glucose Levels Fall

G-Protein Glucose Taken Into Cell

Adenylate Cylase GLUT4 Moved to
Cytoplasm
cAMP
PKA Active Akt Kinase Activation

PK GS PDK1 Activation
Active Inactive
PIP3 Formation
GP-a PI3 Kinase Activation
Active
IRS-1 Activation
Glycogen Broken Down
Tyrosine Kinase Activation
Blood Glucose Levels Rise
Insulin Receptor Pathway
RTKs - Epidermal Growth Factor

Receptor Tyrosine Kinase

Dimerizes on Binding Epidermal Growth Factor (EGF)
Involved in Growth, Proliferation and Cell Differentiation

EGF

EGFR
RTKs - Epidermal Growth Factor

Epidermal Growth Factor Receptor (EGFR)

EGFR Signaling, Part 1 EGFR Dimer

Autophosphorylated
Tyrosines in
Cytoplasmic Domain

Signaling Complex
Assembled on
Phosphotyrosines
GTP

GTP GDP
Prepares Cell for Division
RTKs - Epidermal Growth Factor

Epidermal Growth Factor Receptor (EGFR)

EGFR Signaling, Part 1 EGFR Dimer

Autophosphorylated
Tyrosines in
Cytoplasmic Domain

Signaling Complex
Assembled on
Phosphotyrosines
GTP

GTP GDP
Prepares Cell for Division
RTKs - Epidermal Growth Factor
RAS Activates RAF Kinase

RAF/RAS Activates MEK Kinase

MEK Activates
MAP Kinase Cascade

Transcription Factor
Phosphorylation
Activates Gene Expression
RAS

RAS is a Family of Related Proteins

Each is Monomeric and like the α-subunit of G-Proteins
RAS Proteins Bind Guanine Nucleotides
RAS Swaps GDP for GTP on Activation
RAS Slowly Cleaves GTP to GDP
Human r-RAS

Bound GDP
 RTKs Summary

Dimerization is Important for RTK Activation

RTKs Play Important Roles in Regulating Cell Proliferation
Binding of Ligand Causes Dimerization for Most RTKs
Dimerization Causes Cytoplasmic Tails to Autophosphorylate and Activate
A Signaling Complex Binds to Phosphotyrosines and Communicates Message to
Cell (usually by phosphorylation)
The Insulin Receptor is a RTK that Stimulates Movement of GLUT4 to Membranes
Insulin Signaling Stimulates Phosphoprotein Phosphatase
Phosphoprotein Phosphatase Reverses Effects of Epinephrine
Insulin Signaling Favors Reduced Blood Glucose and Glycogen Synthesis
Epinephrine Signaling Favors Increased Blood Glucose and Glycogen Breakdown
EGFR Dimerizes and Activates on Binding EGF
EGF Signaling Activates Transcription and Favors Cell Division
RAS is Like a G-Protein and Activates Cell Division When Bound to GTP
Turning off EGFR Signaling Involves GTPase (Ras), Phosphatases, and Endocytosis of
Receptors
Steroid Hormone Signaling

Steroid Hormones Control Metabolism, Inflammation, Immune Functions, Water/salt Balance,

Sexual Characteristics, and Response to Illness/Injury
Steroid Signaling Uses Intracellular, Non-membrane Receptors
Five Classes of Steroid Hormones in Two Groups - Corticosteroids and Sex Hormones
Signaling Mostly Affects Gene Expression so Tends to be Slower in its Effects
Steroid Hormone Signaling

Steroid Hormone Released into Blood

Crosses Lipid Bilayer of Target Cell
Binds to Internal Receptor
Internal Receptor Changes Shape,
Becoming Transcription Factor
Transcription Factor Alters Cell’s
Gene Expression
 Steroid Hormone Signaling
5. Hormone-bound
Receptor Binds DNA,
3. Hormone Binds Initiates Transcription
Receptor, Hsp70
2. Movement Across Lipid Bilayer Released
4. Movement of
Hormone-bound
Receptor to Nucleus

1. Hormone Arrives in Blood

2 Receptor Bound to Hsp70

1
5. Transcription
3
Nucleus
4

Cell
Lipid Bilayer
 HSP Release
Hormone Entry Dimerization Movement
to Nucleus
Steroid Hormone Signaling
Transcription
Activation

Glucocorticoid Hormone Signaling

Hormones and Signal Transduction
• Non-Hormone Signaling

Cells Communicate in Other Ways Than With Hormones

Nerve Transmission
Relies on Ion Gradients and Neurotransmitter Molecules to Transmit Signal
Blocked by Ion Channel Blocking Molecules
Prostanoids
Derived from Arachidonic Acid and Exert Effects Near Where They are Released -
Prostaglandins, Prostacyclin and Thromboxanes
Synthesis Inhibited by Steroids and NSAIDs - Aspirin, Ibuprofen

Prostaglandin H2 Thromboxane A2
Signaling Gone Wild
• Signaling Gone Wild

Signaling Proteins Play Important Roles in Growth and Division

Oncogene - A Mutated Gene Whose Activity Can Cause Uncontrolled Growth
Proto-Oncogene - Unmutated Form of an Oncogene
Mutations in Signaling Systems Can Lead to Tumor Formation
Mutations Affecting Protein Structure/Function
Mutations Affecting Expression of Protein
Other Mutations
Hormones and Signal Transduction
• Signaling Gone Wild

Mutations Affecting Protein

Structure/Function

1. GDP Bound RAS Mutated RAS Most Common

RAS Inactive Point Mutation in Cancer

2. GTP Binding Mutated RAS in 90% of

Activates Pancreatic Cancer and
20% of all Cancers
3. GTPase Converts
GTP to GDP, Inactivating
4. Mutations of Amino Acids
11/12 or 61 Inhibit GTPase &
Activate RAS
5. Activated RAS Stimulates Cell Division
 Hormones and Signal Transduction
• Signaling Gone Wild

Not All Tyrosine Kinases are RTKs Src

Src Proteins are Tyrosine Kinases Found in Various
Cell Locations
Dephosphorylated Src Acts to Stimulate Cell Division
Phosphorylation of Src’s Tyrosines Turns it OFF
Mutations that Affect Src’s Phosphorylation Convert
it to an Oncogene
Hormones and Signal Transduction
• Signaling Gone Wild
Mutations Affecting Protein
Structure/Function
Phosphorylated Tyrosines Src
Block Access to its SH2 Domain and
Prevent it From Participating in Signaling
Leaving it Inactive
Src

Mutations Changing These Tyrosines

Leave the Protein Always Activated,
Stimulating Uncontrolled Cell Division
Hormones and Signal Transduction
• Introduction
Mutations Affecting Expression of
Protein
HER2-Herceptin Complex

HER2 Doesn’t Require EGF Binding for Dimerization/Activation

Is Always Signaling Cell to Divide When Dimerized
Mutations Increasing Levels of HER2 Found in Several Cancers
Breast Cancer (15-30%)
Ovarian Cancer
Stomach Cancer
Uterine Cancer
Treated with Monoclonal Antibody - Herceptin
Herceptin Binds HER2’s Extracellular Domain to Prevent Dimerization
 Hormones and Signal Transduction
• Introduction

Other Mutations
Bcr-Abl
Fusion
The bcr-abl fusion links the
tyrosine kinase of abl
with the N-terminus
and transcription control of bcr
bcr bcr-abl fusion

Crossover
22 22/9

All regulation of abl is lost

in the fusion, so the
abl bcr-abl fusion is signaling
‘division’ all the time
9 9/22

Chromosomes 9 & 22 Fusion Chromosomes

Hormones and Signal Transduction
• Introduction
Bcr-Abl
Fusion
Also Known as Philadelphia Translocation
Present in 95% of people with CML (Chronic Myelogenous Leukemia)
Treated with Tyrosine Kinase Inhibitor - Gleevec (Imatinib)

Gleevec has Almost Doubled the Five Year Survival Rate of CML Patients
Other Signaling Considerations

Steroid Hormone Signaling Uses Intracellular, Non-membrane Receptors

Steroid Hormone Receptors Act as Transcription Factors When Bound to Hormone
Non-hormone Signaling Includes Nerve Transmission and Prostanoid Signaling Src Proteins
are Tyrosine Kinases Found in Various Cell Locations
Nerve Transmission Involves Action Potentials Generated by Ion Gradient Changes
Oncogenes Cause Cancer and are Mutated Proto-Oncogenes
Mutations in Signaling Systems Can Lead to Tumor Formation
RAS Mutations that Inhibit GTPase Can Cause Cancer
Mutated RAS Most Common Point Mutation in Cancer
Phosphorylation of Src’s Tyrosines Turns it OFF
Phosphorylated Tyrosines Block Access to Src’s SH2 Domain
Src’s SH2 Domain Controls Access to Other Signaling Proteins
Mutations Changing Src’s Tyrosines Leave the Protein Always Activated
Human EGFR (HER2) HER2 Doesn’t Require EGF Binding for Dimerization/Activation
Overexpression of HER2 Linked to Many Cancers
HER2 Cancers Treated with Herceptin
bcr-abl Fusion links the Tyrosine Kinase of abl with N-terminus & Transcription Control of bcr
bcr-abl Fusions Implicated in Many CMLs
bcr-abl Tumors Fought with Tyrosine Kinase Inhibitor - Gleevec