6. Signaling
• Membranes
Lipid Bilayer Prevents Entry of Most Molecules
Lipid Bilayer
Organization of Bilayer
Around Cell
Information must move across membrane
Signal Transduction
9. 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
Epinephrine
Thyroid Hormone
Epidermal Growth Factor
Progesterone
10. Hormones and Signal Transduction
• Introduction
Cellular Signaling Has Complexity
Responses Aimed at Benefitting Organism
11. Hormones and Signal Transduction
• Binding to Receptor
Steroid Hormones Diffuse Through Membrane
Cytosolic Receptor
Receptor Binding Outside of Cell
Numerous Internal
Message Carriers
Receptor Binding
Inside Cell
Non-steroid Hormone
12. Hormones and Signal Transduction
• Receptors
Interaction of Hormone with Receptor Changes Receptor
Receptor Change Alters Interactions with Other Proteins
Changes on Hormone Binding
13. Hormones and Signal Transduction
• Membrane-bound Receptors
G-Protein Coupled Receptor
14. Hormones and Signal Transduction
G-protein Coupled Receptors (GPCRs)
Almost 800 Genes in Human Genome
460 Olfactory
β-adrenergic Receptor
Membrane Bound
Seven Transmembrane
Domains
Extracellular
Domain
Intracellular
Domain
15. GPCR Action Cycle
Binding of Hormone
Hormone
Structural Change of Receptor
Replacement of GDP by GTP
Activation of an Enzyme
Return to
Original
State
16. 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)
Cyclic AMP (cAMP)
Cyclic GMP (cGMP)
Ca++
Ca++
Ca++
Ca++
Ca++
Ca++
Calcium Ions
17. 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
γ
GPCR
GTP GDP
Resting State
β
γ
GPCR +
β
γ
GPCR
Active
Epinephrine
18. Hormones and Signal Transduction
β-adrenergic Receptor Signaling
ATP
cAMP + PPi
Activated by binding to
α-subunit of G-protein
Second
Messenger
Also Membrane Bound
Adenylate
Cyclase
Transmits Signal
Creation of the Second Messenger
Adenylate
Cyclase
19. Hormones and Signal Transduction
• Actions of the Second Messenger
Protein
Kinase A
(Inactive)
4 cAMP
Activated
Activity
Altered
by
Phosphorylation
Regulatory Subunits
Catalytic Subunits
20. Hormones and Signal Transduction
• G-Protein Coupled Receptors Outline
Receptor
G-Proteins
Protein Kinase A
Kinase Cascade
Turning Signal Off
The Coffee Connection
21. Hormones and Signal Transduction
• β-adrenergic Receptor Signaling
ATP
cAMP
PKA-Reg
PKA
GS-P GS
PK
PK-P
GP-b
P-GP-a
Glycogenx
Glycogenx-1
Glucose-1-phosphate
1
2
3
4
5
5a
6
7
Protein Kinase A
Phosphorylase Kinase
Glycogen Phosphorylase
Glycogen
Synthase
23. Hormones and Signal Transduction
• Turning Off β-adrenergic Receptor Signaling
Turning off the Signaling Pathway
β-adrenergic Receptor
G-Protein
cAMP
Protein Kinase A
24. Hormones and Signal Transduction
• Turning Off β-adrenergic Receptor Signaling
Turning Off β-adrenergic Receptor
Blocks and
Favors Endocytosis
Arrestin
G-Protein
Receptor
Kinase
ATP ADP
Exterior of Cell
Cytoplasm
25. Hormones and Signal Transduction
• β-adrenergic Receptor Signaling
G-protein Inactivation
Auto-regulating
Inherent GTPase Activity
β
γ
Hydrolysis
Phosphate
β
γ
Return to Resting State
Adenylate Cyclase Inactivated - no more cAMP
26. Hormones and Signal Transduction
• β-adrenergic Receptor Signaling
PO4
=
PO4
=
X
28. Hormones and Signal Transduction
• β-adrenergic Receptor Signaling
cAMP
PKA
GS-P
PK-P
P-GP-a
Glycogenx
Glycogenx-1
Glucose-1-phosphate
PO4
=
AMP
Phosphodiesterase is
Inhibited by Caffeine
Drinking Coffee Gives
a Small Boost to
Blood Glucose by
Keeping cAMP Levels
Higher
29. 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
32. P
P
PP
P
P P
P
PP
P
P
Assembly of
Signaling Complex
Signaling Complex
Communicates Message to
Cell (usually by phosphorylation)
SH2 Domains of
Proteins Recognize and
Bind Phosphotyrosines
Receptor Tyrosine Kinases (RTKs)
33. • RTK Signaling Overview
Binding of Hormone to
RTK in Membrane
Receptor
Dimerization
Autophosphorylation
Signaling
Complex
Assembly
Communicate
Message
to Cell
Receptor Tyrosine Kinases (RTKs)
34. RTKs - Insulin Receptor
Unlike Other RTKs, Always a Dimer in Membrane
Binding of Insulin Activates Autophosphorylation of Tails
Binding of
Insulin
Autophos-
phorylation
IRS-1
Activation
PIP3
Formation
PDK1
Activation
Akt Kinase
Activation
Stimulate
GLUT4
Movement to
Cytoplasm
Cells
Uptake
Glucose
PI3 Kinase
Activation
Other
Signaling Pathways
Blood
Glucose
Levels
Fall
Insulin Signaling Also
Activates
Phosphoprotein
Phosphatase
35. Hormones and Signal Transduction
Insulin Receptor Pathway
β- Adrenergic Pathway
G-Protein
Adenylate Cylase
cAMP
PKA Active
PK
Active
GS
Inactive
GP-a
Active
Glycogen Broken Down
Blood Glucose Levels Rise
Tyrosine Kinase Activation
IRS-1 Activation
PI3 Kinase Activation
PIP3 Formation
PDK1 Activation
Akt Kinase Activation
GLUT4 Moved to
Cytoplasm
Phosphoprotein Phosphatase
Activated
Glucose Taken Into Cell
PK, GP-a Inactive GS Active
Blood Glucose Levels Fall
Glycogen Made
36. RTKs - Epidermal Growth Factor
Receptor Tyrosine Kinase
Dimerizes on Binding Epidermal Growth Factor (EGF)
Involved in Growth, Proliferation and Cell Differentiation
EGFR
EGF
37. EGFR Signaling, Part 1
Epidermal Growth Factor Receptor (EGFR)
EGFR Dimer
Autophosphorylated
Tyrosines in
Cytoplasmic Domain
Signaling Complex
Assembled on
Phosphotyrosines
GTP GDP
GTP
Prepares Cell for Division
RTKs - Epidermal Growth Factor
38. EGFR Signaling, Part 1
Epidermal Growth Factor Receptor (EGFR)
EGFR Dimer
Autophosphorylated
Tyrosines in
Cytoplasmic Domain
Signaling Complex
Assembled on
Phosphotyrosines
GTP GDP
GTP
Prepares Cell for Division
RTKs - Epidermal Growth Factor
40. 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
41. 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
42. 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
43. 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
44. Cell
Nucleus
Lipid Bilayer
1
1. Hormone Arrives in Blood
2
2. Movement Across Lipid Bilayer
3
3. Hormone Binds
Receptor, Hsp70
Released
4
4. Movement of
Hormone-bound
Receptor to Nucleus
5. Transcription
5. Hormone-bound
Receptor Binds DNA,
Initiates Transcription
Receptor Bound to Hsp70
Steroid Hormone Signaling
46. 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
47. 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
48. Hormones and Signal Transduction
• Signaling Gone Wild
Mutations Affecting Protein Structure/Function
RAS
1. GDP Bound
RAS Inactive
2. GTP Binding
Activates
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
Mutated RAS Most Common
Point Mutation in Cancer
Mutated RAS in 90% of
Pancreatic Cancer and
20% of all Cancers
49. Hormones and Signal Transduction
• Signaling Gone Wild
Not All Tyrosine Kinases are RTKs
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
Src
50. Hormones and Signal Transduction
• Signaling Gone Wild
Mutations Affecting Protein Structure/Function
Src
Src
Phosphorylated Tyrosines
Block Access to its SH2 Domain and
Prevent it From Participating in Signaling
Leaving it Inactive
Mutations Changing These Tyrosines
Leave the Protein Always Activated,
Stimulating Uncontrolled Cell Division
51. Hormones and Signal Transduction
• Introduction
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
Mutations Affecting Expression of Protein
52. Hormones and Signal Transduction
• Introduction
Other Mutations
Bcr-Abl Fusion
Chromosomes 9 & 22
abl
bcr
22
9
Fusion Chromosomes
9/22
22/9
bcr-abl fusion
Crossover
The bcr-abl fusion links the
tyrosine kinase of abl
with the N-terminus
and transcription control of bcr
All regulation of abl is lost
in the fusion, so the
bcr-abl fusion is signaling
‘division’ all the time
53. 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
54. 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
