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ANAT365 > Module 7 - GPCR > Flashcards

Module 7 - GPCR Flashcards

1
Q

what is the main feature of the GPCR structure

A

7 TM domains

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2
Q

what terminus of GPCR is extracellular?

A

N-terminus

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3
Q

what kind of receptor is oxytocin receptor?

A

GCPR

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4
Q

approximately how many genes are predicted for GPCR in human?

A

800 genes

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5
Q

name 5 things that can bind GPCRs to activate them (LIGAND)

A

light (binds rhodopsin), Ca2+, odours/pheromones, endogenous small molecules, proteins

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6
Q

what part of GPCR are hydrophobic?

A

transmembrane domains

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7
Q

name examples of endogenous small molecules that can bind and activate GPCRs

name four

A

peptides, biogenic amines, lipids, nucleosides

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8
Q

name examples of proteins that can bind and activate GPCRs

3

A

pituitary hormones, interleukins, chemokines

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9
Q

GPCRs are coupled to what kind of protein? what is the structure?

A

heterotrameric G protein made of:
- alpha subunit (binds GTP)
- beta subunit
- y (gamma) subunit

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10
Q

what are the effector for Gprotein that get activated via Galpha subunit vs GBy subunit?

A

Ga = Enzyme, Canal, Transporter
GBy = Enzyme, Canal

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11
Q

what are vaguely the 4 steps of GPCR pathway?

A

activation
desensitization
internalization
recycling

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12
Q

where are GPCR synthesized? where do they go after?

A

synthesized in the ER, then go to Golgi to get further processed and are targetted to the surface via TRANS GOLGI network

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13
Q

how are GPCRs removed from the PM?

A

endocytosis (or caveola?) -> early endosome -> recycled to PM or sorted to lysosomes

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14
Q

what are the 2 faiths of internalized GPCRs?

A
  • recycling back to PM
  • sorted to lysosome for degradation
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15
Q

what end of the golgi apparatus is the receiving end? (where proteins arrive from the ER)

A

cis (cisternae)

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16
Q

where is the golgi apparatus located in the cell?

A

near the nucleus and the microtubule organizing center

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17
Q

from what face of the golgi apparatus do the secretory vesicles arise from?

A

trans face

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18
Q

mostly what kind of vesicles are secreted by the golgi?

A

clathrin vesicles

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19
Q

where can secreted vesicles from the trans golgi go?

A
  • sorting endosomes
  • cell surface
  • lysosomes
  • secretory granules
  • regulated vesicles
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20
Q

What are RAMPs? explain their role quickly

A

Receptor Activity Modifying proteins: proteins that interact with GPCR and affects its function

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21
Q

how RAMP can affect GPCR pharmacology?

A

pharmacology switch: it can make the GPCR bind to a different hormone / neuropeptide

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22
Q

how many RAMPs are in the RAMP family?

A

3

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23
Q

how can RAMP cause a signaling switch of a GPCR?

A

by changing the 2nd messenger of the GPCR

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24
Q

what is the 3rd type of switch that a RAMP can do to GPCR?

A

trafficking switch (change where receptor goes)

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25
Q

what was the first function that RAMPs were discovered for at first?

A

GPCR chaperone

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26
Q

what is CTR?

A

calcitonin receptor

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27
Q

what is CLR? what did they find about it

A

calcitonin receptor-like receptor: stuck in the ER unless they were expressed in a cell line that expressed RAMP1

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28
Q

what does RAMP1 do to CLR?

A

RAMP1 induces CLR transport to the membrane

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29
Q

What happens to CLR when different RAMP bind to it?

A

CLR binds to a different ligand (CGRP vs adrenomedullin 1 or 2)

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30
Q

where is adrenomedullin expressed?

A

in Kidneys

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31
Q

what happens to CTR depending on what RAMP is binds to?

A

can switch from being a calcitonin receptor to a amylin receptor (hormone)

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32
Q

more specifically what does CLR become when is binds RAMP2 vs RAMP3? how does that affect their faith after internalization?

A
  • binds RAMP2 = becomes AM1 (adreno medullin receptor1) gets degraded by lysosome
  • binds RAMP3 = AM2; gets rapidly recycled
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33
Q

what are the 4 functions of RAMPs?

A
  • pharmacology switch
  • signaling switch
  • trafficking switch
  • chaperone
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34
Q

what is GABA?

A

most widespread inhibitory neurotransmitter in the nervous system

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35
Q

what is special about the GPCR GABABr?

A

it has 2 receptor proteins (GABAB1and GABAB2) and both are necessary to relieve GABAB ER retention signal

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36
Q

how are single (not dimerized) GABAB subunit brought back to the ER?

A

via COPI vesicles that recognize their ER retention signal

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37
Q

what happens when they mutated the c-terminus ER retention signal of GABABr?

A

GABABr can be expressed at the cell surface but does not work

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38
Q

what is special about sst5 (somatostatin receptor 5) expression?

A

it is stimulated/regulated, not constitutive

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39
Q

what is somatostatin?

A

neuropeptide that inhibits all hormones that stimulated pituitary hormones secretion

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40
Q

what is the difference between sst2 and sst5?

A
  • sst 2 A is almost completely at the cell surface at rest.
  • sst 5 is mostly intracellular at rest
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41
Q

what is an intracellular pool?

A

pool of receptors that are ready to be expressed but stay intracellular until they get a signal

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42
Q

where does the intracellular pool come from?

A
  1. neo synthesis via secretory pathway or
  2. internalized receptors that were previously at membrane
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43
Q

what does beta 1 adrenergic receptor mediate?

A

adrenaline effect on the cardiovascular system

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44
Q

why is beta 1 adrenergic so studied?

A

to understand GPCR regulation and signaling

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45
Q

what are the 4 stages of GPCR?

A

resting state
stimulation
phosphorylation
desensitization

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46
Q

name subunits that make up the complex that surrounds GPCR (in the slides, example is beta 1 adrenergic)

A

adenylic cyclase (AC), cognate heterodimeric G protein, AKAP (coordinates further downstream signaling)

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47
Q

what happens instantly after the hormone (ligand) binds to a GPCR?

A

G protein alpha subunit dissociates to bind adenylin cyclase (AC)

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48
Q

what happens when G protein alpha subunit binds to AC following hormone binding to GPCR?

A

AC hydrolyzes ATP to form cAMP

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49
Q

hormone -> GPCR -> protein alpha subunit -> AC -> cAMP -> … what are the next steps?

A

cAMP binds RII on AKAP, which releases PKA.
PKA starts phosphorylating.

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50
Q

in the GPCR cascade, what does PKA phosphorylate?

A

many things, including GRK2 (GPCR kinase)

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51
Q

what does GRK2 phosphorylation do?

A

activates GRK2 so it can itself phosphorylate GPCR to stop signalling

52
Q

what part of GPCR are phosphorylated by GRK2? which phosphorylation stops signaling?

A

c terminus and 3rd intracellular loop.
3rd loop stops signaling

53
Q

what does GPCR phosphorylation by GRK2 trigger?

A

B arrestin recruitment

54
Q

what does B arrestin recruitment to the GPCR complex do?

A

complete block of signaling;
G protein alpha subunit detaches from AC, stopping cAMP synthesis, and goes back with beta subunit

55
Q

what are the 3 groups of GRKs?

A
  1. GRK1, GRK7 (Rhodopsine like)
  2. GRK2, GRK3 (bARK1 et bARK2)
  3. GRK4, GRK5 et GRK6
56
Q

where are each different type of GRKs found?

A
  • GRK5 and GRK7 = cytosol at rest, PM when activated
  • GRK1, 4, 6 = always at membrane
  • GRK2, 3 = targeting controlled by Gby and PIP2
57
Q

which GRKs do most of the GRK job and are present in all cells?

A

GRK2 and GRK3

58
Q

what are the 3 domains of a GRK?

A
  • RGS: GPCR binding domains at n-terminus
  • catalytic domain in the middle
  • PH: phospholipid (membrane) binding domain at c-terminus
59
Q

Which side of GRK interacts with Ga, GBy, clathrin, caveolin, GIT (GRK interacting protein) respectively?

A
  • n-terminus (RGS/GPCR binding domain) interacts with Ga, caveolin
  • Catalytic domains interacts with GIT1, PI3K
  • c-terminus (PH/membrane binding) interacts with GBy, clathrin, acidic lipids
60
Q

what can phosphorylate GRK?

A

not just PKA! many other second messenger proteins like PKC, c-Src, Erk1/2, CaM

61
Q

what are the 2 types of GPCR desensitization?

A
  • classical/homologous desensitization = phosphorylation of c-terminus and 3rd intracellular loop via GRK
  • heterologous desensitization = non-specific desensitization via PKC or PKA (not GRK)
62
Q

what is the first kinase to be activated if the GPCR is linked to the itnracellular release of calcium?

A

PKC

63
Q

are GRKs part of the GPCR TM complex?

A

no they are cytoplasmic and only go to membrane when phosphorylated

64
Q

in what cases does heterologous desensitization happen?

A

low hormone levels / low GPCR activation levels, because the first thing activated is PKA and PKC because they are attached to GPCR

65
Q

are GRKs part of the membrane complex?

A

no, they are cytoplasmic and must be recruited

66
Q

which type of desensitization is specific to that receptor?

A

homologous

67
Q

are there more GRK or PKA/PKC phosphorylation sites on GPCR?

A

more GRK site than PKA (homologous desensitization is stronger)

68
Q

why is heterologous desensitization faster than homologous?

A

because PKA is already attached to GPRC, meanwhile GRK must be recruited

69
Q

what happens to GPCR at low agonist (ligand) concentration?

A

GPCR gets phosphorylated by PKA and functionally uncoupled from the G protein

70
Q

what happens to the agonist-induced phosphorylation levels of GPCR under LOW agonist concentration when PKA sites vs GRK sites are missing? both missing? what can we conclude?

A
  • missing PKA sites = decrease by 60%
  • missing GRK sites = decrease by 20%
  • missing both = decrease by like 65%
  • conclude that at low agonist conc, PKA does the phosphorylation
71
Q

what happens to the agonist-induced phosphorylation levels of GPCR under HIGH agonist concentration when PKA sites vs GRK sites are missing?

A
  • PKA missing = decrease by 40%
  • GRK missing = decrease by 50%
  • both = additive effect = decrease by 90%
72
Q

during high agonist concentration, GPCR gets phosphorylated by what?

A

PKA and GRK

73
Q

is recovery of signaling possible under low or high agonist conc?

A

low conc. at high concentrations, B arrestin binds the receptor and no recovery

74
Q

what recruits B arrestin to the GPCR? remember

A

GRK phosphorylation of GPCR

75
Q

what is B arrestin role?

A

terminate GPCR signaling and complete the internalization process

76
Q

which arrestin are ubiquitously expressed? where are the others?

A

2 and 3; the others are only in the retina
(1 = rods; 4 = cones)

77
Q

what are 2nd messenger kinases?

A

PKA/PKC

78
Q

what does each side of arrestin do?

A

N = binds PKA/PKC
C = binds GPCR, clathrin, AP2 (for internalization)

79
Q

how is arrestin recruited?

A

phosphorylation of GPCR by GRK after ligand binding

80
Q

B arrestin limits the magnitude of the B2 AR signal not only through promoting desensitization but also by ?

A

targeting cAMP-inactivating phosphodiesterase enzymes to the activated receptor

81
Q

more specifically how does B arrestin work?

A
  • adaptor of GPCR to clathrin coated pits
  • attracts cAMP-inactivation enzymes such as PDE to inactivate cAMP
82
Q

what are the 2 types of GPCR endocytosis?

A

lipid-raft dependent or clathrin-dependent (also caveolae but no details)

83
Q

what happens after sst2 internalization? how long does it take?

A

re-appearance at cell surface! takes 60 minutes, full recovery can take longer

84
Q

where does sst2 go after being internalized? how did they find out?

A

golgi (TGN) marked by syntaxin-6 (or so they thought)

85
Q

briefly re-explain sst2 pathway of internalization

A

somatostatin binds -> sst2 desensitized -> clathrin-coated pits -> interrnalized -> endosomes -> TGN -> returned to cell surface

86
Q

what is brefeldin A?

A

drug that disperses the TGN

87
Q

what happens to sst2 and syntaxin 6 after addition of brefeldin A?

A

they don’t disperse with the TGN

88
Q

what TGN marker did they use?

A

PIST

89
Q

after finding that brefeldin A does not affect internalized sst2 or syntaxin 6, what did they conclude?

A

Internalized sst2A receptors reside in a syntaxin 6-positive compartment which is distinct from the TGN and the recycling endosome

90
Q

what are GSVs?

A

Glut4 storage vesicles, distinct from TGN

91
Q

what is glut4?

A

insulin receptor that allows for glucose clearing from plasma

92
Q

what are the similarities between GSVs and GSVL (sst2 sequestering compartments)? what similarity is missing?

A
  • Juxta-nuclear localization
  • Syntaxin 6 positivity
  • Distinct from the TGN
  • Brefeldin A insensitivity
  • storage functionality
  • Rab 10 association
    missing to know if GSVL do regulated recycling
93
Q

what was the hypothesis about GSVL recycling?

A

they are recycled in response to hormonal signaling

94
Q

what happened to sst2 when you add hormone (CRF) vs without hormone?

A

adding the hormone increased recycling of SST2a

95
Q

what hormone triggers SST2 recyling to surface?

A

CRF

96
Q

what happens to a c-terminus mutant B arresting?

A

no agonist-promoted internalization of GPCR

97
Q

why do C-terminus mutation of B arrestin stops internalization?

A

because B arrestin can’t bind to the phosphorylated receptor anymore

98
Q

what are class A GPCR?

A

GPCR with short interaction with B arrestin (b arrestin stays at membrane when gpcr is internalized), no co-localization in endosomes, low
affinity for visual arrestin

99
Q

what are class B GPCR?

A

long interaction with B arresting (B arrestin follows inside cell), co-localization in endosomes, high affinity for visual arrestin

100
Q

name class a GPCR and class b

A

A = B2-adrenergic, dopamine D1, endothelin ETa
B = angiotensin AT1A, Neurotensin NTR1, substance P (NK1), oxytocin, vasopressine V2

101
Q

what happens if you inverse C terminal domains (chimera) of B2-adrenergic (class A) and vasopressin V2R (class B)? what does this mean?

A

inverses the internalization properties of the receptors;
class A/B is determined by sequence in C-terminus

102
Q

what is the 3rd role of B-arrestin? (en plus de arrest signal and deactive cAMP)

A

attracting other secondary messenger and creating signaling endosomes

103
Q

what modification of B arrestin triggers the intracellular signal propagation (attracting other 2nd messenger and signalling endosomes)?

A

ubiquitination

104
Q

what are the signaling endosomes created by B arrestin good for?

A

they spatially and temporally extend GPCR signaling - prolongation of GPCR signaling

105
Q

GPCR ubiquitination does what?

A

targeted for degradation by proteasome

106
Q

what is the rationale of the paper?

A

study the intracellular trafficking of a GPCR in this case sst2A somatostatin receptor in vivo following agonist stimulation

107
Q

what is OCT?

A

sst2r agonist

108
Q

what happens to the sst2Arlocalization in the hippocampus after OCT stimulation?

A

moves from dendrites (molecular layer) to the cell bodies (dentate gyrus and stratum pyramidal)

109
Q

where is sst2 in the dentate gyrus before stimulation?

A

in the molecular layer

110
Q

in figure 2, the dark equals what?

A

lots of fluorescence aka lots of sst2A

111
Q

how long does it take for sst2a to fully come back to the molecular layer after OCT stimulation?

A

7 days

112
Q

why do they care about somatostatin?

A

it has inhibitory activity that could be useful for many condition like strokes or epilepsy comes from damage from excitotoxicity

113
Q

why did measure sst2 mRNA levels? what did they find?

A

to see if sst2a was going to the cell body for neosynthesis.
no change in mRNA = does not go to nucleus for neosynthesis

114
Q

if sst2a doesn’t go to the cell body for neosynthesis, why does it?

A

for sequestration and RECYCLING

115
Q

what is CHX?

A

drug that inhibits ribosome

116
Q

what happened to sst2a after cells treated with CHX? what can we conclude?

A

does not affect sst2a internalization.
again shows that sst2a internalization is not for neosynthesis

117
Q

what did they find using immunogold immunocytochemical staining of sst2?

A

that it goes to the golgi after OCT stimulation

118
Q

why did they use confocal microscopy?

A

to prove sst2A goes to TGN using sst2 and golgi/ER markers

119
Q

what markers did they use for ER and cis-golgi?

A

PDI: marker for the endoplasmic reticulum; GM130: cis-Golgi marker

120
Q

did they find colocalization with ER or cis-golgi? (PDI / GM130)

A

no

121
Q

what marker did they use for TGN?

A

syntaxin 6

122
Q

confocal microscopy showed that recycling is over (back to normal) after how long?

A

48h

123
Q

why are the findings not so accurate anymore?

A

syntaxin 6 is not a golgi marker!

124
Q

what did adding colchicine do? conclusion?

A

toxin depolarizes microtubules -> stopped sst2 internalization.
sst2 transport to TGN is microtubule dependent.

125
Q

what is the significance of the study?

A

sst2a is rapidly trafficked ot TGN; likely to induce profound desensitization of sst2a-expressing neurons to SOM;
SOM could help prevent excessive excitation in epilepsy and stroke!

ANAT365 (10 decks)

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