Module 8 - Receptor mediated cell signaling, metabolism

Question 1

Describe the steps of RTK receptor signaling

Answer 1

• growth factor binds to inactive RTK receptor
• receptors dimerize and RTK becomes active
• cytoplasmic tails of RTK are autophosphorylated
• proteins bind to RTK phosphotyrosines
• target proteins are phosphorylated
• downstream signaling pathways are activated

Question 2

Describe the steps of EGF receptor signaling through Ras activation

Answer 2

• EGF binds to EGFR1
• EGFR1 and EGFR2 dimerize and EGFR1 phosphoryltes the EGFR2 Tyr
• conformational change leads to EGFR2 phosphorylating EGFR1 Tyr
• GRB2 adaptor (an SH2 adaptor that also contains SH3) binds to phosphorylated Tyr residues
• SOS (a GEF protein) binds to the SH2/SH3 adaptor
• Ras (a G protein) binds to the SOS adaptor
• GTP exchange for GDP activates Ras downstream signaling pathways
• OR -
• RasGAP stimulates GTPase in Ras and deactivates signaling

Question 3

What is the difference between SH2 and SH3?

Answer 3

SH2 binds pTyr and Asn parts of the receptor

SH3 binds Pro parts of the SOS protein

(having both is what creates the bridge)

Question 4

The Raf-MEK-ERK pathway is a type of…

Answer 4

EGF receptor signaling

Question 5

What are the two kinds of EFG defects and how do they lead to cancer?

Answer 5

dominant gain of function mutations: one affected gene copy in the GTPase domain causes uncontrolled cell growth (oncogenes) by stimulating MAPK and being insensitive to GAP regulation

recessive loss of function mutations: two affected gene copies inactivate tumor suppressors

Question 6

What kind of cancer is commonly associated with Ras mutations? How much? What mutations?

Answer 6

80% of pancreatic cancers

missense mutations G12D, G12V, and G12R which all decrease the GTPase activity of oncogenic Ras protein

Question 7

Regarding GTP binding, GAP binding, and GTPase activity, what combinations of strong and weak produce normal response vs. cancer?

Answer 7

if all are strong, there will be normal response

if GTP is strong but GAP and GTPase are weak, there will be cancer (no regulation, only stimulated growth)

if GTP and GTPase are weak but GAP is strong, there will be cancer but not as bad (less regulation but also less growth)

Question 8

Describe the structure of the insulin receptor

Answer 8

a2B2 tetrameric complex linked by disulfide bonds

Question 9

Describe the insulin receptor signaling pathway through PI-3K activation

Answer 9

• one molecule of insulin binds to the alpha subunit
• conformational changes in the beta subunit activate intracellular kinase domains
• autophosphorylation of tyrosines causes a final conformational change in the beta subunits
• SPLIT:
• one branch activates GRB2, SOS, and Ras for MAPK signaling and altered gene expression and cell division
• Other branch is the Insulin specific pathway (cont. below)
• phosphorylated tyrosine recruits and phosphorylates IRS
• phosphorylated IRS binds to PI-3K
• PI-3K signaling leads to glucose uptake and glycogen synthesis

Question 10

What is the difference in SHC and IRS proteins recruited to the insulin receptor pathway?

Answer 10

SHC: recruited for MAPK pathway, binding domain is Asn-Pro-Gln-pTyr

IRS: recruited for PI-3K pathway, binding domain is Asn-Pro-Ala-pTyr

Question 11

Describe the PI-3K pathway starting with activated PI-3K

Answer 11

• PI-3K phosphorylates PIP2 to PIP3 (terminated by PTEN)
• PIP3 recruits PDK1
• PDK1 phosphorylates Akt
• Akt phosphorylates downstream proteins and causes glucose uptake and glycogen synthesis in liver cells

Question 12

What is the difference between IP3 and PIP3

Answer 12

PIP3 is 3, 4, 5
IP3 is 1, 4, 5

PI-3K phosphorylates PIP2 to PIP3

PLC breaks PIP2 into DAG and IP3

Question 13

How do PDK1 and Akt stay at the membrane?

Answer 13

PDK1 and Akt have pleckstrin homology domains that bind to membrane associated PIP3 lipids in the insulin signaling pathway

Question 14

TNF pathways do not involve any…

Answer 14

kinases

Question 15

TNF receptor signaling initiates two pathways that lead to….
What determines the chosen outcome?

Answer 15

cell death (apoptosis) or cell survival

pathway and outcome is determined by relative abundance of downstream signaling proteins (FADD takes apoptotic pathway, TRAF2 takes cell survival pathway)

Question 16

TNF is mediated by adaptors that have what types of domains?

Answer 16

death domains or death effector domains

Question 17

Describe TNF receptor signaling up to when the two pathways diverge

Answer 17

• 3 molecules of TNF-a bind to the TNF receptor trimer
• binding stimulates the exchange of SODDs for TRADDs (SODD inhibits signaling)
• TRADD proteins initiate adaptor complex formation and downstream signaling (paths diverse at the level of TRADD)

Question 18

Describe the TNF receptor signaling starting when the two pathways diverge

Answer 18

Apoptotic pathway:
- DD and DED complexes assemble
- procaspase 8 autocleaves
- CASP8 cleaves procaspase 3
- CASP3 cleaves cellular proteins leading to cell death

Cell survival pathway:
- NIK/RIP mediate IKK phosphorylation
- IKK phosphorylates IKBa, activating p50/p65
- active p50/p65 form the active NFkB complex which moves to the nucleus
- NFkB increases expression of anti-apoptotic genes that inhibit CASP8 and CASP3 (stop apoptosis pathway)

Question 19

What is the structure of death domains of TNF?

Answer 19

six alpha helices

Question 20

How is CASP3 activated by CASP8?

Answer 20

CASP8 cleaves caspase 3 at Asp175 to a partially active procaspase 3

an inactive procaspase 3 finds another and noncovalent interactions form between the two subunits to form a partially active enzyme

procaspase 3 cleaves at Asp28 and Asp9 (extras off the ends)

two sets of procaspase subunits (p12 and p17) assemble into a heterotetramer to form a fully active CASP3

Question 21

What are nuclear receptors? What are the basic steps?

Answer 21

ligand regulated transcription factors that bind to specific DNA sequences in target genes and recruit coregulatory proteins to module transcriptional initiation rates

• ligand binds
• complex binds DNA
• coregulatory proteins recruited and activate RNA polymerase

Question 22

What are the two types of nuclear receptors and their similarities and differences?

Answer 22

steroid and metabolite receptors

same:
- bind DNA specifically
- recruit regulatory proteins

different:
- DNA sequence is different (steroid bind inverted repeat sequences as homodimers, while metabolite bind direct repeat DNA sequences at heterodimers)
- steroid bind head to head while metabolite bind head to tail

Question 23

If two cell types are exposed to a steroid molecule but only one type had activated gene expression, what is the difference between the two cell types?

Answer 23

only one expresses the cognate steroid receptor

Question 24

Where do the zinc fingers on nuclear receptor homodimers bind?

Answer 24

cysteines in the major groove of DNA

Question 25

Define metabolic flux and what key factors determine the flux

Answer 25

rate at which metabolites are interconverted between reactants and products

• availability of substrate (diet or stored reserves, Q mass action ratio)
• level of enzyme activity (enzyme levels, catalytic activity, compartmentation/location)

Question 26

What is liver cell metabolism like before breakfast?

Answer 26

low insulin, high glucagon

degradation of glycogen to glucose, formation of glucose from gluconeogenesis

low glycolysis of glucose to pyruvate and low synthesis of glycogen from glucose

Question 27

What is liver cell metabolism like after breakfast?

Answer 27

high glucose

synthesis of glycogen from glucose (for storage) and breakdown of glucose by glycolysis into pyruvate (for energy)

low degradation of glycogen to glucose, and low gluconeogenesis of glucose

Question 28

What is the definition of metabolism?

Answer 28

biochemical reactions that turn chemical energy into work

Question 29

In the hierarchy of metabolic pathways, what are the 4 macromolecules, 6 primary metabolites, and 7 small biomolecules?

Answer 29

4 macromolecules: proteins, nucleis acids, carbohydrates, and lipids

6 primary metabolites: amino acids, nucleotides, fatty acids, glucose, pyruvate, and acetyl-CoA

7 small biomolecules: NH4+, CO2, NADH, FADH2, O2, ATP, and H2O

Question 30

How is dG’o calculated?
What does a negative or positive value indicate?

Answer 30

let reaction run until it reaches equilibrium

find concentrations of reactants and products at equilibrium and calculate Keq

plug Keq into dG qeustion and dG=0 (bc. equilibrium) and solve for dG’o

negative indicates the reaction is favorable in the direction written, positive indicates it is favorable in the reverse direction

Question 31

How do the values of dG and dG’o relate to metabolism?

Answer 31

dG is calculated from the mass action ratio (actual concentrations of reactants and products) because things are not at equilibrium in living systems

Question 32

How can unfavorable reactions be used in metabolism?

Answer 32

by linking them to a favorable reaction to create a net negative dG

Question 33

What is beneficial about protein complexes?

Answer 33

they increase the efficiency of coupled reactions through substrate channeling