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Mol. Cell > Midterm No. 3, Opus 3 > Flashcards

Midterm No. 3, Opus 3 Flashcards

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1
Q

In a GPCR path, what does the active receptor protein do?

A

It acts like a GEF

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2
Q

In a GPCR path, what does the active effector protein do?

A

It acts like a GAP

Does GTP –> GDP + pi for Galpha, creates conformational change that causes Galpha to dissociate from the effector, inactivating it

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3
Q

In the example of a basic GPCR that we examined in class, what differentiated the inactive receptor and the active receptor (aside from the bound ligand)?

A

The active receptor had a conformational change in its 7 TMDs

The TMDs were shifted around, altering the protein’s cytoplasmic conformation

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4
Q

In the example of a basic GPCR that we examined in class, what specifically activated the effector?

A

Galpha binding to the effector

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5
Q

In the example of a basic GPCR that we examined in class, how is the signal turned off?

A

The effector acts like a GAP for Galpha, does GTP –> GDP + Pi. This causes a conformational change in Galpha which causes Galpha to dissociate from the effector, inactivating it and resetting the system

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6
Q

In GPCRs, is Galpha always the effector’s activator?

A

Not always

Sometimes Gbeta+Ggamma are the activator

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7
Q

Where do we see Gbeta+Ggamma acting as the effector’s activators?

A

In GPCRs that open/close ion channels

Some scaffolding proteins in RTK/MAPK/Ras cascades

Neurobio stuff

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8
Q

What specific type of mutation would leave the Galpha permanently activated?

A

One that would make it unable to hydrolyze GTP. Galpha always bound to GTP is always active

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9
Q

How would you remove trimeric G proteins from the membrane?

A

They’re lipid-linked to the membrane. A nice salt wash should do it

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10
Q

What enzyme converts ATP to cAMP?

A

Adenylyl (adenylate) cyclase

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11
Q

The most common enzyme for cAMP to interact with is PKA. Describe this interaction

A

PKA exists as 2 catalytic subunits, forms a full 4 subunit molecule

cAMP binding to full 4 subunit PKA and induces a conformational change, causing 2 active subunits to dissociate from the 2 regulatory subunits

The activated PKA subunits can enter the nucleus to phosphorylate CREB, activating it and allowing it to bind to DNA

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12
Q

Does PKA act as a transcription factor?

A

No. It regulates transcription factors (phosphorylates them, turns them on/off)

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13
Q

What is the function of activated PKA?

A

Enters the nucleus and phosphorylates TFs, turns them on/off

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14
Q

What specific type of mutation in PKA’s regulatory subunits would lead to permanently active PKA?

A

There’s more than one right answer

You could make the regulatory subunits unable to bind to the catalytic subunits

You could make cAMP permanently bound to the regulatory subunits

You could delete the regulatory subunits altogether

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15
Q

What specifically activates PKA?

A

cAMP binding to PKA causes it to dissociate into 2 pairs of two subunits

1 pair includes its 2 active subunits (the ones that enter the nucleus and phosphorylate TFs)

1 pair includes its 2 regulatory subunits (these prevented the active subunits from being active)

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16
Q

How many G-alpha proteins do humans have?

A

21

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17
Q

Humans have more G-coupled receptors than G-alphas. Why?

A

This creates higher specificity with ligand signal binding

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18
Q

What does G(alpha)s do?

A

Stimulates adenylyl cyclase

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19
Q

What does G(alpha)i do?

A

Inhibits adenylyl cyclase

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20
Q

What does G(alpha)s do to cAMP levels?

A

Increases cAMP

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21
Q

What does G(alpha)i do to cAMP levels?

A

Decreases cAMP

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22
Q

What specifically does cholera toxin do?

A

It binds to G(alpha)s into an “always on” state, preventing it’s GTP from hydrolyzing to GDP, prevents inactivation

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23
Q

Briefly describe the cause of cholera

A

Cholera toxin prevents G(alpha)s’s GTP from hydrolyzing to GDP, locks it into an always on state

Elevated cAMP and PKA levels turn on the cystic fibrosis ABC transporter (CFTR)

Cl- ions constantly exit the cell through CFTR

Eventually Na+ ions are pushed out, causes mass efflux of water out of the cell

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24
Q

How does cholera toxin enter the intestinal cells?

A

Retrograde endocytosis

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25
How do cells shut off the cAMP signal?
Two possible answers cAMP phosphodiesterase converts cAMP to AMP (by using H2O to add -OH to the offending cyclic oxygen) Receptor desensitization via removal of the receptor from the plasma membrane. GPCR Kinase (GRK) and beta-arrestin work together to turn on the active receptors off and endocytose them
26
How does receptor desensitization work? (phrased another way: how do cells prevent responses to GPCR ligands?)
GRK (GPCR kinase) interacts/binds to the GCPR’s cytosolic tail GRK binds to and hydrolyzes ATP to poly-phosphorylate the GCPR’s cytosolic tail. GRK then dissociates Beta-arrestin (betaARR) binds to the now poly-phosphorylated tail after GRK dissociates betaARR recruits SRC and AP2 (AP2 is an adaptor protein that binds to clathrin) to bind to itself The SRC and AP2 proteins help pull the membrane near where the GPCR is into a vesicle (?) Once the vesicle is secured its sent along to be degraded
27
What does PLC (phospholipase C) do?
Cleaves PIP2 into DAG and IP3
28
What is DAG?
1,2,-diacylglycerol It's the membrane lipid portion of phosphatidylinositol
29
What is IP3?
Inositol-1,4,5-trisphosphate It's the inositol portion of phosphatidylinositol
30
Where is phosphatidylinositol (PI) predominantly found?
In the cytosolic leaflet of the plasma membrane, with its head group (inositol) in the cytoplasm
31
What does IP3 do?
Binds to the cytosolic end of IP3-gated Ca2+ channels in the ER membrane, triggers release of the ER's Ca2+ stores into the cytosol
32
How do cells shut off the PLC --> Ca2+ signal?
Immediately after IP3 is created, a phosphatase chews off one of its 3 Pi’s The newly rendered inositol-1,4-bisphosphate cannot trigger Ca2+ channels
33
Why is the PLC --> Ca2+ signal turned off almost immediately after it's turned on?
To prevent the Ca2+ channels from being open any longer than necessary, which would be a danger to the cell
34
List 3 examples of extracellular ligands for RTKs
Growth factors Insulin Ligands for ephrin receptors (involved in neural axon guidance)
35
What happens once a growth factor ligand binds to its RTK?
RTK is activated, receptors dimerize Dimerization allows the RTK’s cytoplasmic domains cross (auto) phosphorylate
36
Where specifically do RTKs phosphorylate each other?
On cytosolic tyrosine residues
37
What is the function of the RTK dimers' phosphorylated tyrosines?
The phosphates recruit and provide binding sites for other proteins that initiate intracellular pathways
38
What is HER2 overexpression associated with?
Aggressive breast cancer
39
What makes HER2 unique?
It has a funky extracellular conformation. Its shape makes it activated all the time, regardless of whether a ligand is present
40
What makes HER3 unique?
It has a tyrosine kinase, but it's nonfunctional. HER3 needs to heterodimerize to produce a signal
41
Can homodimerized HER3 produce an intracellular signal?
No! HER3 must heterodimerize to produce a signal due to its nonfunctional tyrosine kinase
42
What ligands bind to HER2?
None known (as of now)
43
What receptor family are HER1-4 part of?
RTKs
44
Why is HER2 overexpression associated with cancer?
Because HER2 receptors are hypersensitive to EGF, and thus cells overexpressing HER2 are more likely to proliferate inappropriately
45
How have we used monoclonal antibodies to treat cancer?
We've used a monoclonal antibody that binds to the extracellular DII domain of HER receptors. The antibody prevents dimerization, and also tags the cell for macrophages
46
What have we recently added to the cancer-treating monoclonal antibodies to better optimize them?
We've chemically linked the antibody toa. topoisomerase inhibitor The antibody + inhibitor is internalized via endocytosis, and the inhibitor then enters the nucleus to prevent replication
47
What mutation(s) to an RTK would make it it permanently active/on, independent of any ligands?
If the activation loop was flipped/popped out all the time, then the RTK would be on all the time
48
What mutation(s) to an RTK would amplify its signal?
Duplication of the RTK’s gene Excess transcriptional activators for the RTK gene Decondensed chromatin containing the RTK gene
49
What superfamily are insulin receptors a part of?
RTK superfamily
50
How are insulin receptors different from your average RTK?
Insulin receptors are always dimerized
51
If the insulin receptors are always dimerized, how do they transmit their insulin-ligand signal?
Ligand binding induces a conformational change in the cytosolic domain that brings the tyrosine kinases together, allowing for cross-phosphorylation
52
What's the purpose of a reducing agent in a biochemical experiment?
Reducing agents break protein complexes into subunits by breaking disulfide bonds Can be used to determine presence + placement of disulfide bonds within a protein
53
How large are lipid nanodiscs?
10 - 12 nm in diameter
54
How do the hydrophobic lipids of lipid nanodiscs allow their bound proteins to crystallize for structure determination?
MSP binds lipid nanodiscs together and solubilizes them
55
What is the function of MSP?
To solubilize membrane-bound proteins in lipid nanodiscs
56
What specifically is MSP?
A modified version of ApoA, the protein that binds HDL particles together
57
What oncogenic mutation do almost all human tumors have?
An "always on" mutation in an RTK, Ras, and/or MAPK
58
What is Ras?
A GTPase
59
How is Ras activated?
RTKs and cytokine receptors activate Ras by recruiting GRB2 and Sos to the membrane through specific SH2 and SH3 interactions
60
What is GRB2?
An adaptor protein
61
What is Sos?
A GEF
62
What does Ras do?
It activates a Raf-MEK-MAPK (MAPKKK, MAPKK, MAPK) kinase cascade that activates MAPK activity
63
What does MAPK do?
Phosphorylates transcription factors, turns the TFs on and off
64
What does GRB2 bind to and what domains does it use to do so?
It uses its SH2 domain to bind to one of the active RTK's phosphorylated tyrosines It uses its SH3 domain to bind to Sos (a GEF)
65
What does Sos do?
It's a GEF that activates Ras (in a complex with GRB2) It binds to inactive (GDP) Ras and promotes the dissociation of GDP from Ras GTP then binds to Ras in place of the now removed GDP. This activates Ras Now active, Ras dissociates from Sos
66
How is Ras linked to the membrane?
It's lipid linked (no TMD)
67
What does Ras do once it's activated?
It first dissociates from Sos It then binds to Raf in its N-terminal autoinhibitory domain, activating Raf
68
What is Raf?
A MAPKKK
69
How is Raf activated?
Raf is activated when active Ras binds to its N-terminal autoinhibitory domain
70
In addition to Ras and MEK (MAPKK), what other protein does Raf interact with?
14-3-3
71
What does 14-3-3 do?
It binds to phosphorylated proteins. It inhibits Raf until Ras binds (aka until Raf is activated by Ras)
72
What does active Raf do?
It phosphorylates MEK (a MAPKK) to initiate the kinase cascade
73
What kind of kinase is MAPKKK?
ser/thr kinase
74
What kind of kinase is MAPKK?
Dual specificity kinase ser/thr or tyr
75
What kind of kinase is MAPK?
ser/thr kinase
76
Does MAPK enter the nucleus by itself?
No, it homodimerizes to enter the nucleus
77
Does MAPK phosphorylate only TFs?
No, it can also phosphorylate p90RSK while still in the cytosol
78
What does p90RSK do?
It enters the nucleus to phosphorylate transcription factors, turning them on/off
79
Do MAPK and p90RSK directly alter gene expression?
No, they indirectly alter gene expression by phosphorylating TFs. The TFs are what directly alter gene expression
80
How do scaffolding proteins work?
They are large proteins with slots for specific kinases. Some have kinase activities themselves, acting as one of the kinases in the cascade They separate signaling pathways
80
What's the function scaffolding proteins?
They increase the speed of the cascade and ensure the specificity of kinases involved in kinase cascades The separate signaling pathways
81
How do scaffolding proteins affect the speed of their kinase cascades?
Increases speed
82
How do scaffolding proteins affect the speed of their kinase cascades?
Increases speed
83
How do scaffolding proteins affect the specificity of their kinase cascades?
Increases specificity
84
How do scaffolding proteins affect the amplification of their kinase cascades?
Decreases amplification
85
How can a single ligand have multiple downstream intracellular responses? (RTK)
Many RTK cytosolic tails have multiple phosphorylation sites, and use those multiple sites to recruit multiple binding partners. Each binding partner can start their own kinase cascade / signaling pathway
86
What kind of signaling does Notch function in?
Contact-dependent, juxtacrine signaling
87
Notch: lateral inhibition
Initially a group of cells express both receptor and ligand, the over time the cells differentiate
88
Notch: lateral induction
A receptor cell not next to a ligand cell over time specializes into fate A, but if next to a ligand cell will specialize into fate B
89
A lot of the time, when Notch is involved...
...you'll see two cells next to each other taking on different fates.
90
How is Notch anchored to the plasma membrane?
It's anchored by a single pass TMD
91
What's interesting about Notch receptors containing PEST sequences?
PEST sequences are some kind of target for degradation. Proteins with PEST sequences have a very short half-life
92
The Notch receptor is a bit odd, made of two pieces stuck together. How does this happen?
Like insulin, the receptor starts out a single gene/protein The protein is cut by an enzyme in the golgi The two domains remain attached in a non-covalent Ca2+ dependent manner
93
How are Notch receptor's two domains attached to each other?
They're attached in a non-covalent Ca2+ dependent manner
94
What other protein is the Notch receptor similar to?
Insulin They both start out as a single gene/protein, but are cut by an enzyme in the golgi
95
What happens when a Notch ligand binds to a Notch receptor?
The signal/ligand cell initiates endocytosis of the ligand, which stretches/pulls the receptor The stretching provides access for the receptor to be cut by ADAM 10 Gamma-secretase + nicastrin bind to the Notch receptor stump. Gamma-secretase cuts Notch in its transmembrane domain The intracellular end of the now twice cut Notch receptor has a NLS. the end is now free to go into the nucleus to affect gene transcription
96
What specifically does the Notch receptor bind to?
Delta, a subunit on the end of the membrane-bound Notch ligand
97
What is ADAM 10 and what does it do?
It's a protease It cleaves the Notch receptor in Notch's extracellular domain once the receptor has been stretched by the signal cell's endocytosis
98
How are gamma-secretase and nicastrin related?
Nicastrin is a subunit of gamma-secretase. They're part of a complex together
99
Where does gamma-secretase cut the Notch receptor?
It cuts an alpha helix in Notch's transmembrane domain

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