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CORE II Block 2 > G proteins > Flashcards

G proteins Flashcards

1
Q

Where do G proteins get their name?

A

they bind nucleotides (mainly GTP)

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2
Q

Which type of G-protein is highly involved and mutated in cancer biology?

A

small G proteins (aka monomeric G proteins). e.g. Ras superfamily

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3
Q

Which types of hormones act on G proteins and what are the general responses?

A

glucagon, epinephrine, adrenaline. Cause energy mobilization and oxygen distribution

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4
Q

Describe the signal amplification made possible by G proteins.

A

Each G-protein coupled receptor or tyrosine kinase can activate many G proteins which can activate many effectors and second messengers.

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5
Q

List the two main subfamilies of GTP-binding proteins (G proteins)

A
  1. large, or heterotrimeric
  2. small, or monomeric

(elongation factors are also technically G-proteins, because they bind GTP)

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6
Q

What domain do all G proteins have in common?

A

Ras homology domains. These allow for GTP binding, and have GTPase activity.

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7
Q

What secondary structures are common of G proteins?

A

six beta sheets and 5 alpha helices

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8
Q

Describe the post-translational modiifications that are typical for G proteins and allow for membrane targeting and function.

A
  • prenylation (e.g. farnesylation)
  • myrositoylation
  • palmitoylation
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9
Q

What are the conserved structural motif sequences for G proteins?

A

N/TKXD motif (interacts with GTP)

GXXXXGKS/T motif (interacts with beta and gamma phosphates of GTP), also known as the P-loop (P=phosphate binding)

DXXG and SAK motifs (provide specificity for guanine rather than adenine nucleotides)

switch 1 and switch 2 which bind gamma phosphate of GTP

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10
Q

Describe the switch on/off mechanism for G protein activation/inactivation

A

thr35 of switch 1 and gly60 of switch 2 bind the gamma GTP phosphate, while P-loop holds the rest, enabling a loaded spring conformation of the protein. When the gamma phosphate is hydrolyzed, these interactions break and cause the G protein to become inactive.

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11
Q

Describe the fast reaction step underlying G alpha activation.

A

Ga-GDP (inactive) binds GEF. GEF removes GDP and binds GTP, adding GTP to Ga (active).

GEF can actually bind both GTP and GDP, but here binds GTP because the concentration of GTP/GDP is very high in the cyotplasm.

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12
Q

Describe the inactivation step of G alpha.

A

Ga-GTP (active) is irreversibly hydrolyzed so the gamma phosphate of GTP is removed, leaving behind Ga-GDP (inactive). This hydrolyzation is caused by the intrinsic GTPase activity of the G-protein, but is accelerated by GAPs (GTPase-activated proteins).

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13
Q

What are the GAPs specific to heterotrimeric G proteins?

A

RGS proteins

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14
Q

Which G protein subunits of heterotrimeric G proteins stabilize the inactive form?

A

Gby (beta gamma) stabilize the GDP-bound form

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15
Q

Which G protein modulator helps move G proteins throughout the cell?

A

GDIs (guanine dissociation inhibitors) shield the G protein hydrophobic tail so that GDP does not dissociate and the protein stays inactive as it is recycled between different cellular compartments.

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16
Q

Describe the different interactions of effectors with G proteins.

A
  1. preformed binding site. no conformational change. This is direct binding of the G protein to its effector (e.g. ras G protein to Raf effector)
  2. direct allosteric modulation. G protein binds directly and changes conformation of the effector (e.g. Gs subunit binding to AC, allowing AC to bind ATP). Gi is another one of these G protein subunits
  3. indirect allosteric modulation. G protein binds an inhibitor of the effector, thus changing the activity of the effector (e.g. Gt subunit binds inhibitory domain of phosphodiesterase, so it can go on to degrade cAMP)
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17
Q

Which signaling molecule in the section of material is a transducer?

A

G protein

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18
Q

Which form of the G protein does effectors have a high affinity for?

A

The GTP-bound form. Effectors will bind to the switch domains of GTP-bound G proteins

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19
Q

In what cell type were G proteins discovered?

A

lymphoma cells. Rodbell and Gilman. 1994.

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20
Q

True or false: G proteins only activate pathways regulated by second messengers.

A

False. It activates ERKs and Akt/PKB, both of which are not regulated by second messengers.

21
Q

How can G protein alpha subunits s and i be measured for activity? How is would this assay be the same?

A

The assay would be the same because both act on the same protein, AC. We could measure cAMP to measure stimulation of AC. However, would need phosphodiesterase inhibitors, so that cAMP does not get degraded when we want to measure it.

22
Q

describe Gq/11 G protein subunit.

A

It is an alpha subunit that activates phospholipase c (downstream of tyrosine kinase, makes IP3 and DAG)

23
Q

Describe the G protein alpha subunit G12/13.

A

It links the GPCR to Rho, enhancing its kinase activity

24
Q

Mechanistically, how can an activated GPCR activate G proteins?

A

The cytosolic domain can act as a GEF to remove GDP from alpha subunit of G protein and replace it with GTP.

25
Q

What is the 7TM receptor?

A

It is another name for the GPCR. There are five major classes, including rhodopsin-like, and they all engage G proteins on the third intracellular loop, where GDP is exchanged for GTP.

26
Q

List the ways in which the effects of GPCR activation can be turned off.

A
  1. GTPase activity of G proteins
  2. Increased expression of GAPs (helps GTPase of G proteins)
  3. removal of the agonist from the receptor
  4. second messenger elimination
  5. degradation of GEF/the receptor itself
  6. receptor desensitization/downregulation

(receptor dimerization may also provide additional levels of GPCR regulation)

27
Q

What are the two major pathways by which GPCR’s generate small intracellular mediators.

A

cAMP pathway (e.g. lypolysis)

Ca2+ pathway (e.g. action potential generation)

28
Q

What kinase is activated by cAMP? What are some examples of cellular responses mediated by its activation?

A
  • thyroid hormone synthesis
  • glycogen breakdown
  • heart rate increase and force of contraction
  • progesterone secretion
  • water reabsorption
  • learning and memory
29
Q

How is PKA regulated by PDE?

A

Because PDE degrades cAMP, and PKA is activated by PKA, PKA can therefore be inactivated if PDE is present and active

30
Q

How do GPCRs increase cytosolic Ca2+ levels? Which kinase is activated as a result?

A

G protein is activated and acts on PLC which cleaves PL into DAG and IP3. IP3 acts on IP3R on the ER, allowing calcium to leave the ER into the cytoplasm.

Binding of calcium in cyto to calmodulin allows calmodulin binding to CaM kinase, activating it.

31
Q

How can pertussis toxin be used to study G protein alpha subunit Gi? How does this technique overcome a problem in studying G proteins?

A

Normally, we would remove the component being studied to see what its function is. However, presence of G proteins is essential for cell viability. Instead, petussis toxin ribosylates the terminal portion of Gi so it uncouples from the GPCR. Gi will therefore be bound to GDP indefinitely, as the GEF activity of the receptor cannot replace GDP with GTP. Then measure Gi activity by measuring cAMP in the cell. In the presence of pertussis toxin, we would expect an increase in cAMP (Gi is an inhibitor of AC)

32
Q

Describe how Gq/11 signaling can be studied using cyclic depsipeptide inhibitors. What is the drawback?

A

Cyclic depsipeptide inhibitors bind to the switch domain of the Gq/11 subunit, preventing the exchange of GDP and GTP, effectively inactivating it. In excitatory cells, we would see a result of no contraction due to no calcium being released in the presence of the inhibitor.

Here we measure Ca2+ and IP3 second messengers. This is a drawback because cellular calcium can be changed by many different mechanisms. IP3 is not a good measurement either because PLC can also be activated by the beta gamma G protein subunits.

33
Q

List some diseases associated with G protein defects.

A
  • cancer (consititutive activation of GPCR or small G protein mutations or Gq mutation)
  • endocrine diseases (pseudohypoparathyroidism - Gs mutation)
  • psychiatric disorders (catecholamine and seratonin receptors)
  • cholera and pertussis toxin (ADP ribosylation of Gs and Gi respectively)
  • cardiovascular disorders (hypertension and heart failure - GPCRs coupled to Gq/11 and G12/13)
34
Q

List the 5 monomeric G proteins

A
  1. Ras
  2. Rho
  3. Rab
  4. Arf
  5. Ran (no subfamily)
35
Q

What is Ras G protein involved in?

A

Cell proliferation and differentiation

36
Q

What is G protein Rho involved in?

A

actin cytoskeleton, gene transcription and lipid metabolism

37
Q

What is G protein Rab involved in?

A

vesicle trafficking and transport to target compartment

38
Q

What is G protein Arf involved in?

A

formation of transport vesicle

39
Q

What is G protein Ran involved in?

A

Chromosome dynamics and nuclear import/export. This is the only member of the monomeric G proteins that has no subfamily

40
Q

Which monomeric G protein is highly involved in cancers?

A

Ras

41
Q

Describe the post-translational processing of Ras G protein that allows it to function.

A

In general, Ras is post-translationally modified to be targeted to the cell membrane.

  • Ras is farnesylated to microsome membrane within the cell
  • a methyl is then added to the Ras protein
  • Ras is then palmitoylated and prenylated so it can stick into the plasma membrane, where it functions
42
Q

How might anticancer drugs target dysfunctional Ras?

A

It can prevent its post translational modifications that put it in the cell memrbane. As long as Ras is in the cytoplasm it can be degraded.

43
Q

Describe signaling upstream of Ras G protein.

A
  • tyrosine receptor kinase is activated by ligand, and phosphorylates SHC inside the cytoplasm
  • GRB2 is an adaptor protein linking SHC and GEF.
  • SOS GEF activates Ras, which is bound to the inner leaflet of the cell membrane
44
Q

Describe downstream signaling modulated by Ras G protein.

A
  • activated Ras does not activate any second messengers, but activates effectors like Raf and Akt
  • These play a role in cancer by inhibiting apoptosis and promoting proliferation (Akt normally activates p53 indirectly, a pro-apoptotic protein)
45
Q

Describe some therapeutic agents targeting Ras and Ras-dependent pathways.

A
  • farnesyltransferase inhibitors
  • antisense oligonucleotides against Ras and Raf (effector of Ras)
  • kinase inhibitors targeting Ras effectors
  • kinase inhibitors targeting upstream pathways (like phosphorylation of the tyorsine kinase receptor)
46
Q

Describe activation of Ras signaling in different tumors.

A
  • Ras mutation
  • EGFR overexpression (a type of receptor tyrosine kinase)
  • PTEN loss (A phosphatase that acts on Akt to inhibit it)
  • Akt amplification
  • PI3K amplification (directly amplified by Ras)
47
Q

Why is it hard to target a single monomeric G protein in cancer?

A

Cells can often circumvent treatment, because G proteins do not work in isolation.

Example: Rho/Rac and Arf interact in cytoskeleton arrangement and assembly

(monomeric G proteins can also interact with GPCRs)

48
Q

Which kinase types are effectors of monomeric G proteins?

A

Ser/Thr and Tyr kinases

CORE II Block 2 (6 decks)

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