1. Mechanisms of Hormone
Action

2. General principles: 1. Signals act over different ranges.
2. Signals have different chemical natures.
3. The same signal can induce a
different response in different cells.
4. Cells respond to sets of signals.
5. Receptors relay signals via
intracellular signaling cascades.

3. Cells sense and respond to the environment
External signals are converted to
Internal Responses
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

4. 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.

5. 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. Relay molecules in a signal transduction pathway
Primary
Messenger
Secondary
Messengers
Target
Enzymes
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
Cascade Effect

7. activates 100 control factors
Each protein in a signaling pathway
Amplifies the signal by activating multiple copies of the next component in the pathway
- activates an enzyme activity, processes 100 substrates /second
primary signal
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. 1 102 104 105 106 108 A Signal Cascade amplification
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
A Signal
Cascade
amplification 1
102
104
105
106
108

9. Receptors relay signals via intracellular SIGNALING CASCADES
amplification

10. Main Types of Receptors
ION CHANNEL RECEPTORS
G-PROTEIN-COUPLED RECEPTORS
KINASE-LINKED RECEPTORS
INTRACELLULAR RECEPTORS

11. Cell-surface receptors -large &/or hydrophilic ligands
ion-channel-linked
Trimeric
G-protein-linked
enzyme-linked
(tyrosine kinase)

12. Receptor protein is part of an ion channel protein complex
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. Nerve Cell communication
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: Na+ Cl- - + 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. Intercellular Communication
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.

16. Intercellular Communication
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

18. 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 .

19. Kinases - Phosphorylation
Phosphatase - Dephosphorylation
Tyrosine-OH Tyr-Kinases
Serine-OH Ser/Thr-Kinases
Threonine-OH
„dual specificity“ Kinases

20. 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).

22. Growth hormone receptor
Tetrameric complex constructed in presence of growth hormone GH
GH binding
&
dimerisation OH
Binding
of kinases HO OP PO
ATP
ADP
Activation and
phosphorylation
GH receptors
(no kinase activity) OH HO
kinases
Kinase active site
opened by induced fit
Growth hormone binding site
Kinase active site

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.

24. Intracellular receptors
Chemical messengers must cross cell membrane
Chemical messengers must be hydrophobic
Example-steroids and
steroid receptors
CO2H
H2N
Steroid
binding region
Zinc
DNA binding region
(‘zinc fingers’)
Zinc fingers contain Cys residues (SH)
Allow S-Zn interactions

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

27. 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.

29. 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

30. 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).

32. 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 Gs subunit activates adenylyl cyclase and cAMP production, whereas an inhibitory Gi subunit inhibits adenylyl cyclase and cAMP production.

33. 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.

35. 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.

37. 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).

38. Down-regulation of GPCR/cAMP/PKA Signaling
A number of events contribute to the termination of signaling by a GPCR. These include:
dissociation of the hormone from the receptor,
hydrolysis of GTP by Ga
hydrolysis of cAMP via cAMP phosphodiesterase,
phosphorylation and “desensitization” of receptors by kinases such as PKA and ß-adrenergic receptor kinase (BARK).
In addition, GPCRs can be removed from the membrane by vesicular uptake.

39. G-protein activation “molecular switch” inactive (b) Ligand binds
G-protein associates
(c) GDP-GTP exchange
-Subunit dissociates
Active G-Protein-GTP
-> allosteric modulator
of target effector enzyme
active

40. All G-proteins – similar structure/activation
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

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. Activated G-protein G-protein-GTP activation of
Effector Enzyme adenylyl cyclase produces the 2nd messenger cAMP
Activated
G-protein

43. 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.

44. PKA exists in two different states inside cells
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. Phosphorylase kinase inactive + P active Breaks down Glycogen
Protein Kinase A
Phosphorylates downstream target enzymes
inactive
+ P active
Phosphorylase kinase
Breaks down
Glycogen
Into Glucose

46. What are targets for Protein Kinase A??
cAMP regulated pathways
Function target tissue signal
Glycogen breakdown muscle epinephrine
Glycogen breakdown liver glucagon Heart rate cardiovascular epinephrine
Water reabsorption kidney antidiuretic
hormone

47. How to shut it off? Auto Shut-off G-protein -subunit is on a timer
No ligand
Inherent
GTPase activity
Auto
Shut-off

48. cAMP-phosphodiesterase
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 Gs – 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. PIP2
DAG
Lipid
Soluble
InsP3
Water
Soluble

52. 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 Go(other) and Gq G proteins conduct the signal from the GPCR to PLC. This is the pathway used in a1-adrenergic GPCR signaling in the liver. *

53. 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 Protein Kinase C InsP3 ligand- gated Ca++ channels
Activates
Protein
Kinase C
(Starts
Cascade)
InsP3
Ligand
for ER
ligand-
gated
Ca++
channels
 Ca++ levels

56. Response: 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)

57. 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. .

58. 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.

59. 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.

61. 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.

63. “Second messenger“
DAG, IP and Ca++
10-3 M
10-7 M

64. Metabolism of Diacylglycerol
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