L7: Cell Signaling Flashcards
1
Q
RTK structure characteristics
A
- integral membrane proteins
- extracellular binding domain that binds hormone
- membrane spanning domain
- cytoplasmic tyrosine kinase domain
2
Q
ligand binding induced activation “switch”
A
- dimerization - stabilized active conformation
- INTERchain cross phosphorylation (Autophosphorylation)
- cytoplasmic domain p-Y residues initiate assembly of signaling complexes
- assemble signal effectors with SH2 domains
3
Q
active receptor
A
- scaffold for signaling molecules
4
Q
SH2 domain
A
- phosphotyrosines in proteins
- conserved track in RTK
5
Q
SH3 domains
A
- polyproline tracks in proteins
6
Q
PH domains
A
- acidic membrane surface
7
Q
protein interaction domains
A
- autonomously folding units of ~100 amino acids that have surfaces for binding to proteins or membranes
- complexes formed by these units carry out intracellular signaling at sites where RTKs are localized at the cell membrane
8
Q
RTK Mediated Signaling summary
A
- RTK structure characteristics
- ligand binding induced activation “switch”
- active receptor = scaffold for signaling molecules
- multiple assemblées on the activated receptor simultaneously activate different signal relays
9
Q
Ras activates
A
- activated receptor
- GEF recruitment
10
Q
Raf activates
A
- Ras-GTP binding to negative regulatory domain
11
Q
MAPKK activates
A
- Raf phosphorylation of activation loop
12
Q
MAPK activates
A
- MAPKK phosphorylation of activation loop
13
Q
components of Ras pathway
A
- Ras
- Raf
- MAPKK
- MAPK
14
Q
PI3K pathway components
A
- PI3K
- PKB
15
Q
PI3K
A
- activated receptor binds PI3K
- binding promotes allosteric changes that activate the effector enzyme
16
Q
PBK
A
- Akt
- recruitment to localized PIP3 produced by PI3K
17
Q
GPCR Mediated signaling summary
A
- G - coupled receptors
- heterotrimeric G- proteins
- Activated G-proteins regulate 2nd messenger system
18
Q
G-coupled protein receptors
A
- extracellular region
- integral membrane proteins
- seven transmembrane segments
- cytoplasmic tail
- noncovalently bind GTP or GDP
19
Q
heterotrimeric G-proteins subunits
A
- G alpha
- G beta
- G gamma
20
Q
G alpha
A
- GTPase
21
Q
G beta and G gamma
A
- always in a complex
22
Q
Receptor activation stimulates
A
- G alpha to exchange GDP for GTP
- G alpha - GTP to dissociate from G beta gamma
23
Q
G protein - G alpha s
Effector enzyme
2nd messenger
Protein Kinase
A
- G alpha s
- increase adenylate cyclase
- cAMP (cAMP is made from ATP)
- increases PKA
24
Q
G protein - G alpha q
Effector enzyme
2nd messenger
Protein Kinase
A
- G alpha q
- increase phospholipase C beta (splits PIP2 into IP3 and DAG)
- DAG, IP3 (Ca2+)
- IP3 releases calcium from ER which helps activate PKC
- Increase PKC
25
G protein - G alpha i
Effector enzyme
2nd messenger
Protein Kinase
- G alpha_i
- decrease adenylate cyclase
- cAMP
- decrease PKC
26
G protein - G alpha I
Effector enzyme
2nd messenger
Protein Kinase
- G alpha I
- increase phospholipase C
- DAG, IP3 (CA2+)
- increase PKC
27
what does adenylate cyclase do
- catalyzes the synthesis of cAMP
| - free catalytic subunits phosphorylate key target proteins
28
what does phospholipase C do?
- catalyzes synthesis of IP3 and DAG
- IP3 binds to receptors on ER - releases Ca2+
- DAG and Ca2+ activate PKC
- PKC phosphorylates key target proteins
29
GAP
- GTPAse activating protein
| - results in GTP hydrolysis and Ras inactivation
30
stimulus
- hormones
- cytokines
- neurotransmitters
- light
- mechanical stress
31
receptors
- bind stimulus with high specificity
32
transduction
- information transfer through chemical relays between cellular proteins with on/off switches
33
cellular responses
- changes in
- gene expression
- enzyme activities
- proliferative capacity
- survival/apoptosis
- cell shape - cytoskeleton impacted
- motility
34
two key types of cell surface receptors
- receptor tyrosine kinase
| - g-protein coupled receptors
35
hormones and growth factors acting through RTK
- insulin
- nerve growth factor
- epidermal growth factor, fibroblast growth factor, platelet-derived growth factor
36
3 major receptor tyrosine kinase signaling pathways
- Res-Map Kinase
- PI3 Kinase
- Phospholipase Cy
37
Ras-MAP Kinase
- GTPase and protein phosphorylation
38
PI3 kinase
- lipid phosphorylation
39
phospholipase Cy
- phospholipid second messengers
40
PI3K subunits
- can be activated simultaneously with the RAS pathway
- has p85 regulatory subunit
- SH2 domains recruited to RTKs
41
PI3K 2nd messenger
- PI3K recruited to autophosphorylated RTKs at SH2
- PI3k phosphorylates PIP2 to make PIP3
- PKB recruited to PIP3 at plextrin homology domain
42
multiple replays coordinate activities required for cellular response to stimulus
- one side recruits Ras MAPK pathway which results in cell proliferation
- other side recruits PI3K pathway which results in cell survival genes
43
physiological roles of G protein coupled receptors
- vision (opsin)
- smell (olfactory receptors)
- taste (GCPRs in taste cells)
- ANS neurotransmission (blood pressure, heart rate, etc)
44
Importance of Receptors and Cell Signaling
1. Cell communication
2. Long-distance signalling (hormones/nutrients)
3. Short-distance signaling (development)
45
Agonist
activates signalling
46
Antagonist
inhibits signalling
47
Name 4 types of receptors
1. Neurotransmitter receptors
2. G-protein coupled receptors
3. Tyrosine Kinase receptors
4. Nuclear receptors
48
Role of second messengers
Mediate signaling
ex: cAMP, cGMP, Calcium ions, IP3 and DAG
49
Function of Tyrosine Kinase Receptors (TKR)
Their kinase activity phosphorylates tyrosine residues on target proteins - these phosphorylations change protein function and can trigger a signaling cascade
58 human TKRs
Often used involved in growth control
Ex: Insulin, FGR, EGFR, VEGF, PDGF, etc.
50
Symptoms of T2D
high blood sugar levels, high insulin levels, weight loss, fatigue, increased hunger/thirst, acanthosis nigricans
51
Acanthosis nigricans
darkened skin disorder associated with diabetes
52
Rabson-Mendehall syndrome
results from mutations in the insulin receptor, resulting in severe insulin resistance with dental and skin abnormalities
53
Function of AKT in insulin signaling
Insulin signaling works through AKT (protein kinase B) that phosphorylates multiple proteins to control metabolism
54
What does insulin signaling promote (metabolically)?
1. glucose uptake by trafficking transporters to the membrane
2. glycolysis
3. protein synthesis
4. synthesis of glycogen and fatty acids
(inhibits gluconeogenesis)
55
Structure of insulin
Protein hormone consisting of two chains connected by disulfide bonds
56
Structure of insulin receptor
Tyrosine kinase receptor present in the membrane
- homodimer: each chain consists of two subunits linked by a disulfide bond
- alpha-subunit extracellular
- beta-subunit transmembrane and intracellular
57
How is the insulin receptor unusual for RTKs?
it is a "pre-formed" homodimer, meaning that it is a dimer before binding to insulin.
Most other RTKs dimerize only after binding ligand.
58
What is the effect of insulin binding to the IR?
After binding insulin, a conformational change results in the receptor becoming autophosphorylated on several tyrosine (of beta-subunit), leading, to a second conformational change and activation of its kinase domain to phosphorylate other proteins.
59
What is the role of the activation loop in the IR?
the activation loop normally blocks the kinase domain, but autophosphorylation of the beta-subunits causes conformational change where activation loop swings out of the way so the kinase domain is open to phosphorylate additional proteins.
60
Detail the insulin signaling cascade.
When hormone binds, a conformational change triggers trans-autophosphorylation of the 𝛽-subunits and a subsequent conformational change that causes the phosphorylated receptor, now activated, to bind insulin receptor substrate protein (IRS-1) and phosphorylate it at multiple tyrosines. Signaling proteins bind IRS-1 on its phosphorylated tyrosines and activate signaling pathways. One protein that binds IRS-1 is phosphoinositide 3-kinase that converts PIP2 to PIP3ƻ and PIP3 results in activation of the PDK1 kinase. Akt (Protein kinase B) is phosphorylated and activated by PDK1 and it phosphorylates additional downstream proteins.
bottom line: phosphorylated and activated Akt that can phosphorylate others
61
Metformin
Drug used to treat T2D.
Reduces GNG, increases sensitivity to insulin signaling and decreases absorption of glucose from food.
62
Function of EGF
Epidermal Growth Factor
controls epidermal cell division and binds to an RTK
small protein stabilized by disulfide bonds
63
EGF Receptor Dimerization
In the absence of ligand, the EGF receptor (EGFR) exists as monomers.
There is a "dimerization arm" on the extracellular domain of each monomer that mediates dimerization upon growth factor binding.
In the absence of growth factor, the dimerization ar is bound to a site on the monomer so that it cannot form dimers.
Binding of EGF causes a conformational change that releases the dimerization arm so the monomers can dimerize.
Each monomer binds EGF.
64
Dimerization of EGFR leads to
autophosphorylation of the EGFR
65
Explain the EGFR signaling pathway
Upon ligand binding, EGFR undergoes a conformational change that brings the C-terminal region of one monomer into the kinase domain of the other, resulting in cross auto-phosphorylation.
The phosphorylated EGFR binds to the Grb2 adaptor protein that has an SH2 domain and 2 SH3 domains.
The SH2 domain of Grb2 binds to the phosphorylated tyrosines on EGFR.
The SH3 domains bind to Sos. Sos is a guaning-exchange protein that interacts with the Ras signaling protein.
Sos exchanges GTP for GDP on Ras, and the Ras-GTP becomes an active signaling component.
66
What is the function of activated Ras in the EGF Signaling pathway?
Activated Ras activates a signaling cascade.
67
Detail the signaling cascade after Ras activation.
Ras is a small G-protein that has GTPase activity. In its active GTP-bound form, Ras interacts with the Raf protein kinase and activates it. Raf phosphorylates and activates the MEK kinase, which then phosphorylates and activates the ERK kinase. Erk phosphorylates numerous target proteins that stimulate cell division and other cellular functions; these targets include transcription factors.
68
What are some Ras-like proteins?
the Ras, Rho, Arf, Rab and Ran families
69
When is Ras signaling terminated?
By phosphatases that inactivate the active, phosphorylated proteins, including EGFR.
Ras has GTPase activity that inactivates itself over time by converting GTP to GDP -- this process can be accelerated by the presence of GTPase-activating proteins (GAPs).
70
Nuclear Receptor function
transcription factors that bind DNA upon ligand binding
ligands are often lipid-soluble compounds that can diffuse through the membrane
71
What is cortisol?
- glucocorticoid steroid hormone produced in the adrenal gland that increases blood sugar (liver GNG) and suppresses the immune system
- released during the day and during stress or low blood sugar
72
Glucocorticoid Receptor (GR) structure
GR is a transcription factor with a DNA binding domain (DBD), transcriptional activation domain, (TAD), ligand-binding domain, and nuclear localization sequence (NLS).
73
Explain how ligand-binding activates the GR as a TF.
GR is normally present in the cytoplasm bound to a chaperone (HSP), which masks the NLS and keeps the GR in the cytoplasm.
When the ligand (cortisol) enters the cell, it binds GR and displaces the HSP, resulting in a conformational change.
The conformational change unmasks the NLS.
GR forms a homodimer, and the homodimer translocates to the nucleus.
The GR dimer binds to DNA at enhancers (GRE - glucocorticoid response element) on target genes.
The transcriptional activation domain (TAD) recruits coactivators.
The GR-coactivator complex binds general/basal TFs and activates transcription.
74
Name lipophilic hormones that bind nuclear receptors
(1) steroid hormone -- cortisol, estrogen, aldosterone
(2) thyroid hormone
(3) retinoic acid (vit. A)
(4) Vitamin D
75
HIF-1alpha
- an example of a nuclear receptor; belongs to basic-loop-helix-PAS (bHlH-PAS) transcription factors
- responds to low oxygen levels
- activates the transcriptional response to counteract low oxygen conditions
- important to cancer growth
76
HIF-1alpha under normoxia
HIF-1alpha undergoes proteasomal degradation under normal oxygen conditions.
77
HIF-1alpha under hypoxia
HIF-1alpha enters the nucleus and binds to DNA (HREs) to promote transcription.
78
What are G-Protein Coupled Receptors (GPCRs)?
Membrane receptors that possess 7 transmembrane (TM) domains.
They include olfactory receptors, neurotransmitter receptors and light receptors.
Each GPCR binds a specific ligand
79
What type of receptor does Glucagon bind?
GPCR
80
What molecules are involved in Glucagon signaling?
Glucagon signaling involves a GPCR, cAMP and PKA cascade.
PKS phosphorylates multiple proteins that control metabolism.
81
Metabolic actions of Glucagon signaling
Promotes: breakdown f glycogen and triglycerides to use as fuel (glucose, fatty acids), GNG, and AA uptake by liver
Inhibits: glycolysis in the liver
82
GPCR signaling with G-proteins
GPCRs signal through G-proteins, which have alpha, beta and gamma subunits.
In the unstimulated state, G𝛼 is bound to GDP and G𝛼 and G𝛾 are associated with the plasma membrane. When stimulated, the GPCR undergoes a conformational change tha causes an exchange of GDP for GTP in G𝛼. G𝛼-GTP itself undergoes a large conformational change resulting in dissociation from the receptor and from G𝛽 and G𝛾. G𝛼-GTP activates adenylate cyclase that results in the formation of cAMP and activation of protein kinase A.
83
Adenylate cyclase
Membrane protein with cytoplasmic domains that have adenylate cyclase activity.
When activated by Gas-GTP, adenylate cyclase converts ATP to cAMP.
84
Describe Signal Termination with the GPCR
cAMP inactivation
Gas-GTP inactivation
GPCR inactivation.
85
cAMP degradation
occurs via cAMP phosphodiesterase (degrades cAMP to AMP)
ensures cAMP levels are kept low in unstimulated cells and helps terminate the reaction
86
G𝛼 Inactivation
Over time, G𝛼 inactivates itself by hydrolyzing its GTP (i.e. G𝛼 is a slow GTPase – so signaling has time to take place). G𝛼-GDP dissociates from adenylate cyclase and reforms the trimeric G-protein complex so it can interact with another GPCR.
87
GPCR Inactivation
Multiple mechanisms:
(1) GPCR signaling is reduced when the ligand or hormone dissociates.
(2) During signaling, a kinase is activated that phosphorylates the GPCR and reduces its signaling activity.
(3) Phosphorylated receptor is recognized by Arrestin that further reduces activity and GPCR signaling ability.
88
Do all GPCRs activate G-proteins?
No!
Some receptors activate stimulatory G-proteins (Gs-𝛼) that increase cAMP levels (e.g. epinephrine, glucagon, adrenocorticotropic hormone (ACTH)). Other receptors activate inhibitory G-proteins (Gi-𝛼) that reduce cAMP levels (e.g. prostaglandin E1 ƑPGE1)ƹ adenosine).
