Lecture 35: Cell Signaling II

Question 1

Enzyme-linked cell surface receptor features

Answer 1

• Ligand binding activates intrinsic enzymatic activity
• Very high affinity (endocrine, paracrine)
• Termination by receptor-mediated endocytosis
• RTKs, receptor Ser/Thr kinases

Question 2

Receptor Tyr Kinase (RTK) process

Answer 2

1. Ligand binding → dimerization and kinase activation
2. Auto-cross-phosphorylation of Tyr residues (active)
3. Binding/activation of signaling proteins (effector, adaptor, coupling)
4. Cascade initiation

Question 3

MAP Kinase cascade

Answer 3

1. Auto-Pi → adaptor binds
2. Adaptor binds Ras activating protein (GEF)
3. RasAP activates RAS by GEF activity
4. Active Ras triggers MAP Kinases cascade (Ser/Thr, Thr/Tyr, Ser/Thr)
5. MAP kinase Pi’s effector proteins and many other things
6. Changes in gene expression + cytosolic/membrane protein activities

Question 4

Receptor Ser/Thr kinase mechanism

Answer 4

2 homodimers (Type I/II) form active tetramer
1. Auto-Pi of constitutively active Type II kinase
2. Ligand binds Type I + II
3. Trans-Pi of Ser/Thr activates Type I dimer
4. SMAD binds + gets Pi from Type I → unfolding + activation
5. SMAD dissociates and dimerizes w/ other subtype
6. Exposure of nuclear localization signal → nuclear translocation

Question 5

Cytokine receptor features

Answer 5

• Ligand binding → assoc. + activation of cytoplasmic enzymes, esp. Tyr kinases
• No intrinsic enzyme activity on receptor
• Paracrine/autocrine
• Terminate by receptor mediated endocytosis and protein phosphatases

Question 6

JAK/STAT pathway (cytokine receptor)

Answer 6

1. Cytokine ligand binding → subunit association
2. JAK activation → cross-Pi → subunit Pi
3. STATs bind
4. Pi + activation of STATs
5. STAT dissociation + dimerization → NLS exposure and nuclear translocation

Question 7

How does Ebola block the JAK/STAT IFN gamma pathway?

Answer 7

Ebola vp24 competes w/ STAT1 dimer for importin (importin responsible for nuclear translocation via Nup), suppressing activation of innate antiviral immunity.

Question 8

JAK/STAT

Answer 8

JAK = Janus Kinase
STAT = Signal Transducer and Activator of Transcription
Example of cytokine receptor

Question 9

Receptor overview

Answer 9

Question 10

Classes of 2nd messengers

Answer 10

Intracellular signals
1. Ions e.g. Ca2+
2. H2O soluble e.g. cAMP, cGMP, IP3
3. Membrane-assoc. e.g. DAG, arachidonic acid

Question 11

Ca2+ as a 2nd messenger

Answer 11

Ca2+ is simply moved around/out of cell (not made/destroyed); local [Ca2+] = detected signal. Balance of Ca-ON and Ca-OFF mechanisms.
Low Ca indicates a quiescent cell.

Question 12

Ca-OFF mechanisms

Answer 12

Mechanisms that remove Ca2+, lowering local concentration
1. Plasma membrane pumps (NCX exchanger, PMCATPase)
2. Sarcoplasmic/Endoplasmic Reticulum pumps (SERCA pumps)

Question 13

Na+/Ca++ exchanger (NCX)

Answer 13

Exchanges Na+ downhill into cell for Ca++ uphill out of cell. Has low Ca++ affinity but high transport rate; bulk transport role.

Question 14

Plasma Membrane Calcium ATPase (PMCA)

Answer 14

Has high Ca++ affinity but low transport rate; fine tuning of Ca++ concentration.

Question 15

Ca-ON mechanisms

Answer 15

Increase Ca concentration
1. Ligand/voltage gated ion channels (plasma membrane)
2. Ryanodine Receptor RyR (SR)
3. IP3 receptor (ER)
RyR/Ip3 examples of Ca-induced Ca release

Question 16

Cyclic nucleotide 2nd messengers

Answer 16

H2O soluble, made and destroyed e.g. cAMP, cGMP

Question 17

cAMP generation

Answer 17

Adenylate cyclase = plasma membrane enzyme w/ 2 catalytic domains. Activation by GPCR G protein aligns domains and increases cAMP production 100X from ATP

Question 18

cAMP destruction

Answer 18

cAMP phosphodiesterase converts cAMP to AMP

Question 19

cAMP activation of PKA

Answer 19

Primary effector mechanism for cAMP. PKA activation separates catalytic subunits from regulatory subunits, allowing nuclear translocation and Pi of CREBs to alter gene expression

Question 20

CREBs

Answer 20

cAMP Response Element Binding Protein. Alter gene expression in response to phosphorylation by PKA, which is activated by cAMP

Question 21

cAMP level regulation

Answer 21

GPCR I and II bind different ligands and have opposed stimulatory/inhibitory activity. Balance of these determines cAMP levels and thus activation profile

Question 22

How does cholera toxin affect cAMP?

Answer 22

Attaches an ADP ribose to G-stimulatory α subunit preventing GTPase activity; increased cAMP leads to excessive water loss

Question 23

How does pertussis toxin affect cAMP?

Answer 23

Attaches ADP ribose to G-inhibitory α subunit preventing G-i activation; increased cAMP alters hormone activity via PKA (high insulin/histamine sensitivity) leading to low glucose/BP

Question 24

cGMP generation/destruction

Answer 24

Guanylate cyclase generates cGMP from GTP
cGMP phosphodiesterase turns cGMP to GMP (has negative feedback)

Question 25

NO stimulation of cGMP production

Answer 25

1. NO ligand binds soluble guanylate cyclase heme iron
2. αβ catalytic domains align leading to greatly increased cGMP production mainly activating PKG

Question 26

Hormonal stimulation of cGMP production

Answer 26

Hormones (e.g. ANP) bind Membrane-Assoc. Guanylate Cyclase; enzyme-linked receptor.

Question 27

Phospholipid derivatives as 2nd messengers

Answer 27

Derivatives of membrane phospholipids can act as 2nd messengers, e.g. IP3, DAG, arachidonic acid, PIP3