1. Chapter 14 Signal-Transduction Pathways
G proteins
Signal-transduction circuits in biological systems have molecular on-off switches that, like those in a computer chip, transmit information when “on”. Common among these circuits are those including G proteins, which transmit a signal when bound to GTP and are silent when bound to GDP

2. Outline
14.1 Heterotrimeric G proteins transmit signals and reset themselves 14.2 Insulin signaling: Phosphorylation cascades are central to many signal-transduction processes 14.3 EGF signaling: signal-transduction systems are poised to respond 14.4 Many elements recur with variation in different signal-transduction pathways 14.5 Defects in signal-transduction pathways can lead to cancer and other diseases

3. Signal Transduction Pathways
• Signal Transduction – chain of events that converts the message “this molecule is present” to a physiological response
Adrenal glands
β-cells in pancreas
Epinephrine腎上腺素
insulin胰島素
Epinephrine
insulin
Epidermal growth factor (EGF) + + +
β-Adrenergic receptor
Insulin receptor
EGF receptor
Energy-store
mobilization
Increased glucose
Uptake/ glycogen storage
Expression of growth promoting gene
(wound repair)
Fig 14.1 Three signal –transduction pathways. The binding of signaling molecules to their receptors initiates pathways that lead to important physiological response

4. Key Steps in Signal Transduction
Release of the primary messenger
Reception of the primary messenger
Delivery of the message inside the cell by the second messenger
Activation of effectors that directly alter the physiological response
Termination of the Signal
Signal
hormone
Feedback pathways
Reception
cell-surface receptor
Amplification
Transduction
Response(s)
Fig 14.2 Principles of signal transduction

5. Common Second Messengers
Second messengers are intracellular molecules
relay information from the receptor-ligand complex
change in concentration in response to environmental signals
Mediate the next step in the molecular information circuit
Signal may be amplified
Often free to diffuse throughout cell
Common second messengers may create “cross talk”
Cyclic AMP
Fig 14.3 common second messenger

6. 14.1 Heterotrimeric G Proteins Transmit Signals and Reset Themselves
Epinephrine
A hormone secreted by the adrenal glands of mammals in response to internal and external stressor
Signaling begins with ligand (Epinephrine) bind to β-adrenergic receptor (β-AR)
A member of seven-transmembrane-helix (7TM) receptors
>20,000 receptors
serpentine (蜿蜒的,蛇的) receptors: the single polypeptide chain "snakes" through the membrane seven times
Fig 14.4 The 7TM receptor

7. 14.1 Heterotrimeric G Proteins Transmit Signals and Reset Themselves
Rhodopsin (Chapter 33)
A well-characterized member of the 7TM receptor family
retina (視網膜) protein
senses the presence of photons (ligand)
initiate the signaling cascade responsible for visual sensation
lysine residue within rhodopsin is covalently modified by a form of vitamin A 11-cis-retinal
exposure to light induces the isomerization of 11-cis-retinal to its all trans form, producing structural changes in the receptor
initiation of an action potential as visual stimulus
Fig 14.5 (A) Structures of rhodopsin

8. β2-adrenergic receptor
Identified the structure by the inhibitor, carazolol, competes with epinephrine
Similarities with rhodopsin
Mechanism
Binding of a ligand from outside the cell induces a structural rearrangement in the part of the 7TM receptor that is positioned inside the cell
Fig 14.5 (B) Structures of β2-adrenergic receptor

9. Ligand Binding to 7TM Receptors Leads to the Activation of heterotrimeric G Proteins
Epinephrine bind to β2-adrenergic receptor
conformational change of cytoplasmic domain
activated G protein
activate adenylate cyclase
catalyzed the conversion of ATP into cyclic AMP (cAMP)
activate Protein kinase A
G proteins, also known as guanine nucleotide-binding proteins
Fig 14.6 Activation of protein kinase A by a G-protein pathway

10. G Proteins Cycle Between GDP- and GTP-Bound Forms
Unactivated state: G protein is bound to GDP
Heterotrimer consisting of α, β, and γ subunits
α Subunit (Gα) binds the nucleotide
A member of the P-loop NTPase family
Participate in nucleotide binding
α and γ subunit anchor to the membrane
Hormone-bound receptor is catalyze the exchange of GTP for bound GDP
GTP binding induces conformational change
Decreases Gα affinity for Gβγ Gα dissociation
Gα can now bind to other proteins (adenylate cyclase)
 Gαs (s =stimulatory)
Fig 14.7 A heterrtrimeric G protein
Because signal through G protein
7TM receptors are called G-protein coupled receptors (GPCRs)

11. Gα binding to adenylate cyclase
a membrane protein that contains 12 membrane-spanning helices
two large cytoplasmic domains form the catalytic part of the enzyme
Converts ATP into cAMP
Fig 14.8 Adenylate cyclase activation
Catalytic fragment

12. What does the cAMP do?
Increase concentration of cAMP can effect a wild range of cellular processes:
cAMP stimulates production of ATP for muscle contraction
Enhance the degradation of fuel stores
Increase the secretion of acid by gastric mucosa
Leads to the dispersion of melanin pigment granules
Diminishes the aggregation of blood platelets
Induces the opening of chloride channels
Most effect of cAMP in eukaryotic cells are mediated by the activation of a single protein kinase: protein kinase A (PKA)

13. Protein kinase A
Consists two regulatory (R) chain and two catalytic (C) chain (Ch10)
In the absence of cAMP inactive
cAMP binds to the R chains releases the catalytic chains  active
Activated PKA then phosphorylates specific serine and threonine in many targets to alter their activity
PKA phosphorylates two enzymes that lead to the breakdown of glycogen (Ch21)
PKA stimulates the expression of specific genes by phosphorylating a transcriptional activator called cAMP response element binding (CREB) protein

14. Epinephrine signaling pathway
On binding of ligand, the receptor activates a G protein that in turn activates the enzyme adenylate cyclase.
Adenylate cyclase generates the second messenger cAMP.
The increase in cAMP results in a biochemical response to the initial signal
Fig 14.9 Epinephrine signaling pathway

15. How is the signal initiated by epinephrine switched off?
G proteins spontaneously reset themselves through GTP Hydrolysis
Intrinsic GTPase activity of Gα -- hydrolyze bound GTP to GDP and Pi.
the bound GTP acts as a built-in clock that spontaneously resets the Gα subunit after a short time period.
After GTP hydrolysis and release the Pi, the GDP-bound form of Gα then reassociates with Gβγ to re form the inactive heterotrimeric protein.
Fig Reseting Gα.
Intrinsic GTPase activity
heterotrimeric protein

16. How is the signal initiated by epinephrine switched off?
G proteins spontaneously reset themselves through GTP Hydrolysis
Signal termination
Hormone dissociates, returning the receptor to its initial, unactivated state
the hormone-receptor complex activates a kinase that phosphorylates serine and threonine residues in the carboxyl-terminal tail of the receptor.
Diminishes the ability to activate G protein
β-adrenergic receptor kinase
Fig Signal termination.

17. Some 7TM receptors activate the phosphoinositide cascade
angiotensin II (血管緊縮素) (ligand, peptide hormone, control blood pressure) -- angiotensin II receptor
angiotensin II receptor activates Gαq protein
GTP-form Gαq binds to activate the β isoform of phospholipase C
Catalyzes the cleavage of PIP2 into inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG) which stays in the membrane
Fig phospholipase C reaction.

18. inositol 1,4,5-triphosphate (IP3)
soluble and diffuses away from the membrane
causes the rapid release of Ca2+ from intracellular stores in the endoplasmic reticulum
specific IP3 -gated Ca2+ -channel proteins in the ER membrane open to allow calcium ions to flow from the ER into the cytoplasm
Elevated level of cytoplasmic Ca2+ triggers smooth muscle contraction, glycogen breakdown, and vesicle release
Calcium is also a signal molecule: it can bind proteins called calmodulin and enzymes such as protein kinase C
Fig phosphoinositide cascade .

19. Diacylglycerol (DAG) remains in the plasma membrane
activates protein kinase C (PKC)
phosphorylates serine and threonine residues in many target proteins.
the specialized DAG binding domains of this kinase require bound calcium.
IP3 increases the Ca2+ concentration, and Ca2+ facilitates the DAG-mediated activation of protein kinase C.
Fig phosphoinositide cascade .
Both IP3 and DAG act transiently because they are converted into other species by phosphorylation or other processes.

20. Ca2+ is a widely used second messenger
Ca2+ participates in many signaling processes, the properties:
Fleeting changes in [Ca2+] are readily detected
intracellular levels are low to prevent the precipitation of carboxylated and phosphorylated compounds
can bind tightly to proteins and induce substantial structural rearrangements
bind well to negatively charged oxygen atoms (side chains of glutamate and aspartate) and uncharged oxygen atoms (main-chain carbonyl groups and side-chain oxygen atoms from glutamine and asparagine).
Ca2+ can coordinated multiple ligands -from six to eight oxygen atoms- crosslink different protein segments and induce significant conformational changes
Fig calcium-binding site.

21. Scientists can monitor cellular [Ca2+] in real
Fura-2 (molecular-imaging agents) can bind Ca2+ and change their fluorescent properties on Ca2+ binding
Fura-2 binds Ca2+ through appropriately positioned oxygen atoms (in red) within its structure
When Fura-2 introduced into cells, change in available Ca2+ by detection changes in fluorescence
Fig calcium imaging
Red: high Ca2+

22. Calcium ion often activates the regulatory protein calmodulin
Calmodulin (CaM)
a 17-kd protein with four Ca2+-binding sites
Member of EF-hand protein family
EF-hand is a Ca2+ binding motif: Helix, loop, helix motif
7 seven oxygen are coordinated to each Ca2+
serves as a calcium sensor in nearly all eukaryotic cells
At cytoplasmic concentrations above about 500 nM, Ca2+ binds to and activates calmodulin
Conformational changes expose hydrophobic residues
Newly exposed surfaces can bind other proteins, eg, CaM kinase I, and stimulating them
Fig EF hands
Calmodulin-dependent protein kinase
Fig calmodulin binds to the α helices.

23. Signal transduction pathway:
Secondary messenger is increased : Ca2+
The signal is sensed by a second-messenger-binding protein: calmodulin
Second-messenger-binding protein acts to generate changes in enzyme : calmodulin-dependent kinases
Phosphorylate many different proteins
Regulate fuel metabolism
Ionic permeability
Neurotransmitter synthesis
Neurotransmitter release

24. 14.2 Insulin signaling: Phosphorylation cascades are central to many signal-transduction processes
Signal transduction pathways initiated by receptors
kinases as part of the receptor structures
Insulin signaling
Focus on “ leads to the mobilization of glucose transporters to the cell surface”
Insulin
The hormone released in response to increased blood- glucose levels
a peptide hormone that consists of two chains, linked by three disulfide bonds
Fig insulin structure

25. Insulin signaling Insulin receptor
Dimer of two identical subunits (homodimer)
Each unit consists of one chain and one chain linked to one another by disulfide bond
Two α subunits move together around insulin
Each β subunit has a kinase domain similar to PKA
Differs from PKA (protein kinase A)
Is a tyrosine kinase
The kinase is in an inactive conformation when the domain is not covalently modified
Requires tyr in activation loop be phosphorylated
Fig The insulin receptor

26. Insulin binding results in the cross-phosphorylation and activation of the insulin receptor
How is the activation loop phosphorylated?
the two α subunits move together to surround an insulin molecule, the kinase domains also draw closer together
the two β subunits forced together, the kinase domain catalyze the phosphoryl groups from ATP to tyrosine residues in the activation loops -- conformational change takes place – active conformation
Fig Activation of the insulin receptor by phosphorylation

27. Activated insulin-receptor kinase initiates a kinase cascade
Insulin-receptor kinase phosphorylation sites act as docking sites for other substrates, including insulin-receptor substrates (IRS)
IRS-1 and IRS-2 are two homologous proteins with a common modular structure
pleckstrin homology domain: binds phosphoinositide lipids
phosphotyrosine-binding domain
anchoring the IRS protein to the insulin receptor and the membrane
Tyr-X-X-Met (YXXM): four sequences are phosphorylated by the insulin-receptor tyrosine kinase
Fig the modular structure of insulin- receptor substrates IRS-1 and IRS-2

28. IRS act as adaptor protein!!
(not enzyme but serve to tether the downstream components of this signaling pathway to the membrane, eg., phosphoinositide 3-kinase)
Fig insulin signaling
Fig insulin signaling pathway

29. lipid kinases have Src homology 2 (SH2) domains
Src homology 2 (SH2) domains bind to specific phosphotyrosine domains in the IRS protein
Lipid kinases (also called phosphoinositide 3-kinase; PI3Ks ) function on phosphorylate PIP2 to PIP3
Fig Structure of the SH2 domain
Fig Action of a lipid kinase in insulin signaling

30. PDK1 activates another protein kinase Akt
PIP3 activates a protein kinase PDK1 (with a pleckstrin homology domain that specific for PIP3)
PDK1 activates another protein kinase Akt
Akt not membrane anchored, and moves through the cell to phosphorylates targets
control the trafficking of the glucose receptor GLUT4 to the cell surface
target enzymes that stimulate glycogen synthesis

31. Insulin signaling pathway

32. Effect of insulin on glucose uptake and metabolism
Insulin binds to its receptor, which in turn starts many protein activation cascades, include:
translocation of Glut-4 transporter to the plasma membrane and influx of glucose
Glycogen synthesis
Glycolysis
fatty acid synthesis

33. Insulin signaling is terminated by the action of phosphatases
In insulin signaling, three classes of enzymes are of particular importance in shutting off the signaling pathway:
Protein tyrosine phosphatases: remove phosphoryl groups from tyrosine residues on the insulin receptor and the IRS adaptor proteins
Lipid phosphatases: hydrolyze PIP3 to PIP2
Protein serine phosphatases: remove phosphoryl groups from activated protein kinases such as Akt

34. 14.3 EGF signaling: signal-transduction systems are poised to respond
Signal molecule epidermal growth factor (EGF) binds to a receptor tyrosine kinase (EGF receptor; EGFR) that participates in cross-phosphorylation reactions
Epidermal growth factor (EGF)
a 6-kd polypeptide that stimulates the growth of epidermal and epithelial cells
Fig Structure of epidermal growth factor

35. EGF receptor A dimer of two identical subunit
Exits as monomers until they bind EGF
EGF-binding domain that lies outside the cell
Dimerization is mediated by a dimerization arm
the dimer binds two ligand molecules
a single transmembrane helix-forming region
the intracellular tyrosine kinase domain
participates in cross-phosphorylation reactions
One unit phosphorylated by another unit within a dimer
Not within the activation loop of kinase
the tyrosine-rich domain at the carboxyl terminus
5 tyrosines are phosphorylated
Binding of EGF to the extracellular domain causes the receptor to dimerize and undergo cross-phosphorylation and activation
Fig Molecular structure of EGF receptor
Fig EGF receptor dimerization

36. Why doesn’t the receptor dimerized and signal in the absence of EGF?
Conformational different
The dimerization arm binds to a domain within the same monomer
Hold the receptor in a closed configuration
makes it unavailable for interaction with the other receptor
Fig Structure of the unactivated EGF receptor

37. EGF signaling leads to the activation of Ras, a small G protein
After EGF receptor phosphorylation
The SH2 domain of an adaptor protein, Grb-2, binds to the phosphotyrosine residues of the EGF receptor
Grb-2 binds Sos using two Src homology 3 (SH3) domains
SH3 domains bind proline-rich polypeptides
Sos, in turn, binds to Ras and activates it
Ras in a class of proteins called “small G proteins”
GDP-Ras  GTP-Ras
Sos as a guanine-nucleotide-exchange factor (GEF)
Structure of Grb-2, an Adaptor Protein
Fig Ras activation mechanism

38. Activated Ras initiates a protein kinase cascade
GDP-Ras  GTP-Ras change conformation
Binds other proteins, including Raf, a protein kinase (protein-protein interaction)
Raf then undergoes a conformational change that activates its kinase domain
Ras and Raf are anchored to membrane, through a covalently bound isoprene lipid
Raf phosphorylates other proteins, including the kinases termed MEKs
MEKs activate “extracellular signal-regulated kinases” (ERKs)
ERKS phosphorylate many substrates, including other kinases and transcription factors

39. Ras and Raf Signal Transduction
(or other extracellular signaling molecule)
Protein tyrosine kinase domain
Dimer
activated
adapter
phosphorylation
MAPK/ERK kinase
phosphorylation
phosphorylation
Extracellular-signal-
regulated kinase
phosphorylation
Enhanced transcription
More cell division
Fig 12.36

40. EGF signaling pathway
Fig EGF signaling pathway

41. EGF signaling is terminated by protein phosphatases and the intrinsic GTPase activity of Ras
protein phosphatases play key roles in the termination of EGF signaling
Signal activation also initiates signal termination
Ras possesses intrinsic GTPase activity
The activated GTP form of Ras spontaneously converts into the inactive GDP form
GTPase-activating proteins (GAPs) interact with small G proteins in the GTP-bound form and facilitate GTP hydrolysis

42. Small G proteins or small GTPases
Difference between small G proteins and heterotrimeric G proteins
small G proteins
heterotrimeric G proteins
Size
20-25 kd
30-35 kd
Monomer
trimer
Small G proteins have many key mechanistic and structural motifs in common with the Gα subunit of the heterotrimeric G proteins

43. The GTPase cycle
RAS-family proteins are low-molecular-weight guanine-nucleotide-binding proteins.
inactive when bound to GDP
active when bound to GTP
Regulation of this molecular: through a GDP-GTP cycle
guanine nucleotide-exchange factors (GEFs)
catalyse the exchange of GDP for GTP
GTPase-activating proteins (GAPs)
increase the rate of GTP hydrolysis to GDP
In the case of RHO proteins, another layer of regulation is provided by RHO–GDP-dissociation inhibitors (RHOGDIs), which sequester RHO away from the GDP–GTP cycle.
GTPases interact with various effector proteins, which influence the activity and/or localization of these effectors; this ultimately influences cell-cycle progression
Inactive form
Active form
Nature Reviews Molecular Cell Biology 5, (May 2004)

44. RAS signaling
Nature Reviews Molecular Cell Biology 13, (January 2012)

45. 14.4 Many elements recur with variation in different signal-transduction pathways
Signal transduction has many common themes
Protein kinases are central to many signal-transduction pathways
protein kinases often phosphorylate multiple substrates and able to generate a diversity of responses
Second messengers participate in many signal-transduction pathways
Specialized domains that mediate specific interactions are present in many signaling proteins
pleckstrin homology domain: facilitate protein interactions with the lipid PIP3
SH2 domain: mediate interactions with polypeptides containing phosphorylated tyrosine residues
SH3 domain: interact with peptide sequences that contain multiple proline residues

46. 14.5 Defects in signal-transduction pathways can lead to cancer and other diseases
Signal transduction pathways can malfunction, leading to disease such as cancer
For example:
Rous sarcoma virus is a retrovirus that cause sarcoma in chicken
v-src genes is necessary for viral replication
an oncogene and v-Src is a protein tyrosine kinase
Normal chicken muscle cell: c-src
Not induce cell transform proto-oncogene
Encoded a signal transduction protein: regulates cell growth

47. c-Src v-Src Tyrosine residue near the C-terminal
When phosphorylated, it bound intramolecularly by the SH2
the linker between the SH2 domain and the protein kinase domain is bound by the SH3 domain
These interactions hold the kinase domain in an inactive conformation
v-Src
C terminal 19 amino acids are replaced (lacks tyrosine)
Always active and promote unregulated cell growth

48. When Things Go Wrong… Not all oncogenes caused by viruses
A mutated form of Ras can cause cancer
Usually mutations lead to loss of hydrolysis activity
If GTP cannot be hydrolyzed to GDP, stuck in the “on” position, stimulating cell growth
Mutations of “tumor suppressor genes” can cause cancer
E.g., mutations in genes that code for phosphatases involved in EGF signal termination
EGF signaling persists initiated, stimulating inappropriate cell growth

49. Monoclonal antibodies can be used to inhibit signal-transduction pathways activated in tumors
Mutated/overexpessed receptor tyrosine kinases often observed in tumors
Epidermal-growth-factor receptor (EGFR) is overexpressed in some human epithelial cancers, including breast, ovarian, and colorectal cancer
Some EGFR can dimerize and send growth signal even in absence of EGF
Cetuximab (Erbitux) an antibody used therapeutically to prevent dimerization in colorectal cancer
EGFR family member, Her2 overexpressed in~30% of breast cancers
Trastuzumab (Herceptin) an antibody used to inhibit breast cancer

50. Protein kinase inhibitors may be effective anticancer drugs
Protein kinase inhibitors as anticancer drugs
Chronic myelogenous leukemia (CML,慢性骨髓性白血病) often due to chromosomal defect where parts of chromosomes 9 and 22 are translocated, causing overexpression of a kinase (Bcr-Abl kinase)
An inhibitor specific for this kinase, Gleevec (STI-571, imatinib mesylate) very effective treatment
Fig The formation of the bcr-abl gene by translocation

51. DNA Damage and Repair
慢性骨髓性白血病 (chronic myelogenous leukemia) 的病生理機轉最重要的就是第9對以及第22對染色體轉位，t(9:22)，又稱為費城染色體(Philadelphia Chromosome)。
這種染色體轉位會造成原來位在第9對染色體的Abelson (ABL) proto-oncogene接到第22對染色體的breakpoint cluster region (BCR) 基因上，形成BCR-ABL chimeric 基因。正常的ABL 基因在轉錄轉譯後產生的tyrosine kinase會受到嚴密的調控；但發生費城染色體所形成的BCR-ABL fusion基因則失去正常的調控機轉，造成tyrosine kinase過度表現的情形。在慢性骨髓性白血病患者，有大於 90% 的患者會有費城染色體，有大於 95% 的患者可以利用PCR的方式找到BCR-ABL 基因。

52. Cholera霍亂and whooping cough百日咳are due to altered G-protein activity
Chloera toxin- Chloeragen is secreted by the intestinal bacterium Vibrio cholerae
Two functional unit– α β subunit that binds to GM1 ganglisoides of the intestinal epithelium
Catalytic A subunit that enters the cells
A subunits catalyzes the covalent modification of a Gαs protein
The α subunit is modified by the attachment of an ADP-ribose to an arginine
Stabilizes the GTP-bound form of Gαs  active form
continuously activates protein kinase A
opens a chloride channel and inhibit sodium absorption
whooping cough – pertussis toxin from Bordetella pertussis
Adds an ADP-ribose moiety to Gαi, inhibit adenylate cyclase, close Ca2+ channel, open K+ channel, function “off”