Module 7 - GPCR Flashcards
(125 cards)
1
Q
what is the main feature of the GPCR structure
A
7 TM domains
2
Q
what terminus of GPCR is extracellular?
A
N-terminus
3
Q
what kind of receptor is oxytocin receptor?
A
GCPR
4
Q
approximately how many genes are predicted for GPCR in human?
A
800 genes
5
Q
name 5 things that can bind GPCRs to activate them (LIGAND)
A
light (binds rhodopsin), Ca2+, odours/pheromones, endogenous small molecules, proteins
6
Q
what part of GPCR are hydrophobic?
A
transmembrane domains
7
Q
name examples of endogenous small molecules that can bind and activate GPCRs
name four
A
peptides, biogenic amines, lipids, nucleosides
8
Q
name examples of proteins that can bind and activate GPCRs
3
A
pituitary hormones, interleukins, chemokines
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
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
11
Q
what are vaguely the 4 steps of GPCR pathway?
A
activation
desensitization
internalization
recycling
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
13
Q
how are GPCRs removed from the PM?
A
endocytosis (or caveola?) -> early endosome -> recycled to PM or sorted to lysosomes
14
Q
what are the 2 faiths of internalized GPCRs?
A
- recycling back to PM
- sorted to lysosome for degradation
15
Q
what end of the golgi apparatus is the receiving end? (where proteins arrive from the ER)
A
cis (cisternae)
16
Q
where is the golgi apparatus located in the cell?
A
near the nucleus and the microtubule organizing center
17
Q
from what face of the golgi apparatus do the secretory vesicles arise from?
A
trans face
18
Q
mostly what kind of vesicles are secreted by the golgi?
A
clathrin vesicles
19
Q
where can secreted vesicles from the trans golgi go?
A
- sorting endosomes
- cell surface
- lysosomes
- secretory granules
- regulated vesicles
20
Q
What are RAMPs? explain their role quickly
A
Receptor Activity Modifying proteins: proteins that interact with GPCR and affects its function
21
Q
how RAMP can affect GPCR pharmacology?
A
pharmacology switch: it can make the GPCR bind to a different hormone / neuropeptide
22
Q
how many RAMPs are in the RAMP family?
A
3
23
Q
how can RAMP cause a signaling switch of a GPCR?
A
by changing the 2nd messenger of the GPCR
24
Q
what is the 3rd type of switch that a RAMP can do to GPCR?
A
trafficking switch (change where receptor goes)
25
what was the first function that RAMPs were discovered for at first?
GPCR chaperone
26
what is CTR?
calcitonin receptor
27
what is CLR? what did they find about it
calcitonin receptor-like receptor: stuck in the ER unless they were expressed in a cell line that expressed RAMP1
28
what does RAMP1 do to CLR?
RAMP1 induces CLR transport to the membrane
29
What happens to CLR when different RAMP bind to it?
CLR binds to a different ligand (CGRP vs adrenomedullin 1 or 2)
30
where is adrenomedullin expressed?
in Kidneys
31
what happens to CTR depending on what RAMP is binds to?
can switch from being a calcitonin receptor to a amylin receptor (hormone)
32
more specifically what does CLR become when is binds RAMP2 vs RAMP3? how does that affect their faith after internalization?
- binds RAMP2 = becomes AM1 (adreno medullin receptor1) gets degraded by lysosome
- binds RAMP3 = AM2; gets rapidly recycled
33
what are the 4 functions of RAMPs?
- pharmacology switch
- signaling switch
- trafficking switch
- chaperone
34
what is GABA?
most widespread inhibitory neurotransmitter in the nervous system
35
what is special about the GPCR GABABr?
it has 2 receptor proteins (GABAB1and GABAB2) and both are necessary to relieve GABAB ER retention signal
36
how are single (not dimerized) GABAB subunit brought back to the ER?
via COPI vesicles that recognize their ER retention signal
37
what happens when they mutated the c-terminus ER retention signal of GABABr?
GABABr can be expressed at the cell surface but does not work
38
what is special about sst5 (somatostatin receptor 5) expression?
it is stimulated/regulated, not constitutive
39
what is somatostatin?
neuropeptide that inhibits all hormones that stimulated pituitary hormones secretion
40
what is the difference between sst2 and sst5?
- sst 2 A is almost completely at the cell surface at rest.
- sst 5 is mostly intracellular at rest
41
what is an intracellular pool?
pool of receptors that are ready to be expressed but stay intracellular until they get a signal
42
where does the intracellular pool come from?
1. neo synthesis via secretory pathway or
2. internalized receptors that were previously at membrane
43
what does beta 1 adrenergic receptor mediate?
adrenaline effect on the cardiovascular system
44
why is beta 1 adrenergic so studied?
to understand GPCR regulation and signaling
45
what are the 4 stages of GPCR?
resting state
stimulation
phosphorylation
desensitization
46
name subunits that make up the complex that surrounds GPCR (in the slides, example is beta 1 adrenergic)
adenylic cyclase (AC), cognate heterodimeric G protein, AKAP (coordinates further downstream signaling)
47
what happens instantly after the hormone (ligand) binds to a GPCR?
G protein alpha subunit dissociates to bind adenylin cyclase (AC)
48
what happens when G protein alpha subunit binds to AC following hormone binding to GPCR?
AC hydrolyzes ATP to form cAMP
49
hormone -> GPCR -> protein alpha subunit -> AC -> cAMP -> ... what are the next steps?
cAMP binds RII on AKAP, which releases PKA.
PKA starts phosphorylating.
50
in the GPCR cascade, what does PKA phosphorylate?
many things, including GRK2 (GPCR kinase)
51
what does GRK2 phosphorylation do?
activates GRK2 so it can itself phosphorylate GPCR to stop signalling
52
what part of GPCR are phosphorylated by GRK2? which phosphorylation stops signaling?
c terminus and 3rd intracellular loop.
3rd loop stops signaling
53
what does GPCR phosphorylation by GRK2 trigger?
B arrestin recruitment
54
what does B arrestin recruitment to the GPCR complex do?
complete block of signaling;
G protein alpha subunit detaches from AC, stopping cAMP synthesis, and goes back with beta subunit
55
what are the 3 groups of GRKs?
1. GRK1, GRK7 (Rhodopsine like)
2. GRK2, GRK3 (bARK1 et bARK2)
3. GRK4, GRK5 et GRK6
56
where are each different type of GRKs found?
- GRK5 and GRK7 = cytosol at rest, PM when activated
- GRK1, 4, 6 = always at membrane
- GRK2, 3 = targeting controlled by Gby and PIP2
57
which GRKs do most of the GRK job and are present in all cells?
GRK2 and GRK3
58
what are the 3 domains of a GRK?
- RGS: GPCR binding domains at n-terminus
- catalytic domain in the middle
- PH: phospholipid (membrane) binding domain at c-terminus
59
Which side of GRK interacts with Ga, GBy, clathrin, caveolin, GIT (GRK interacting protein) respectively?
- 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
what can phosphorylate GRK?
not just PKA! many other second messenger proteins like PKC, c-Src, Erk1/2, CaM
61
what are the 2 types of GPCR desensitization?
- 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
what is the first kinase to be activated if the GPCR is linked to the itnracellular release of calcium?
PKC
63
are GRKs part of the GPCR TM complex?
no they are cytoplasmic and only go to membrane when phosphorylated
64
in what cases does heterologous desensitization happen?
low hormone levels / low GPCR activation levels, because the first thing activated is PKA and PKC because they are attached to GPCR
65
are GRKs part of the membrane complex?
no, they are cytoplasmic and must be recruited
66
which type of desensitization is specific to that receptor?
homologous
67
are there more GRK or PKA/PKC phosphorylation sites on GPCR?
more GRK site than PKA (homologous desensitization is stronger)
68
why is heterologous desensitization faster than homologous?
because PKA is already attached to GPRC, meanwhile GRK must be recruited
69
what happens to GPCR at low agonist (ligand) concentration?
GPCR gets phosphorylated by PKA and functionally uncoupled from the G protein
70
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?
- 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
what happens to the agonist-induced phosphorylation levels of GPCR under HIGH agonist concentration when PKA sites vs GRK sites are missing?
- PKA missing = decrease by 40%
- GRK missing = decrease by 50%
- both = additive effect = decrease by 90%
72
during high agonist concentration, GPCR gets phosphorylated by what?
PKA and GRK
73
is recovery of signaling possible under low or high agonist conc?
low conc. at high concentrations, B arrestin binds the receptor and no recovery
74
what recruits B arrestin to the GPCR? remember
GRK phosphorylation of GPCR
75
what is B arrestin role?
terminate GPCR signaling and complete the internalization process
76
which arrestin are ubiquitously expressed? where are the others?
2 and 3; the others are only in the retina
(1 = rods; 4 = cones)
77
what are 2nd messenger kinases?
PKA/PKC
78
what does each side of arrestin do?
N = binds PKA/PKC
C = binds GPCR, clathrin, AP2 (for internalization)
79
how is arrestin recruited?
phosphorylation of GPCR by GRK after ligand binding
80
B arrestin limits the magnitude of the B2 AR signal not only through promoting desensitization but also by ?
targeting cAMP-inactivating phosphodiesterase enzymes to the activated receptor
81
more specifically how does B arrestin work?
- adaptor of GPCR to clathrin coated pits
- attracts cAMP-inactivation enzymes such as PDE to inactivate cAMP
82
what are the 2 types of GPCR endocytosis?
lipid-raft dependent or clathrin-dependent (also caveolae but no details)
83
what happens after sst2 internalization? how long does it take?
re-appearance at cell surface! takes 60 minutes, full recovery can take longer
84
where does sst2 go after being internalized? how did they find out?
golgi (TGN) marked by syntaxin-6 (or so they thought)
85
briefly re-explain sst2 pathway of internalization
somatostatin binds -> sst2 desensitized -> clathrin-coated pits -> interrnalized -> endosomes -> TGN -> returned to cell surface
86
what is brefeldin A?
drug that disperses the TGN
87
what happens to sst2 and syntaxin 6 after addition of brefeldin A?
they don't disperse with the TGN
88
what TGN marker did they use?
PIST
89
after finding that brefeldin A does not affect internalized sst2 or syntaxin 6, what did they conclude?
Internalized sst2A receptors reside in a syntaxin 6-positive compartment which is distinct from the TGN and the recycling endosome
90
what are GSVs?
Glut4 storage vesicles, distinct from TGN
91
what is glut4?
insulin receptor that allows for glucose clearing from plasma
92
what are the similarities between GSVs and GSVL (sst2 sequestering compartments)? what similarity is missing?
- 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
what was the hypothesis about GSVL recycling?
they are recycled in response to hormonal signaling
94
what happened to sst2 when you add hormone (CRF) vs without hormone?
adding the hormone increased recycling of SST2a
95
what hormone triggers SST2 recyling to surface?
CRF
96
what happens to a c-terminus mutant B arresting?
no agonist-promoted internalization of GPCR
97
why do C-terminus mutation of B arrestin stops internalization?
because B arrestin can't bind to the phosphorylated receptor anymore
98
what are class A GPCR?
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
what are class B GPCR?
long interaction with B arresting (B arrestin follows inside cell), co-localization in endosomes, high affinity for visual arrestin
100
name class a GPCR and class b
A = B2-adrenergic, dopamine D1, endothelin ETa
B = angiotensin AT1A, Neurotensin NTR1, substance P (NK1), oxytocin, vasopressine V2
101
what happens if you inverse C terminal domains (chimera) of B2-adrenergic (class A) and vasopressin V2R (class B)? what does this mean?
inverses the internalization properties of the receptors;
class A/B is determined by sequence in C-terminus
102
what is the 3rd role of B-arrestin? (en plus de arrest signal and deactive cAMP)
attracting other secondary messenger and creating signaling endosomes
103
what modification of B arrestin triggers the intracellular signal propagation (attracting other 2nd messenger and signalling endosomes)?
ubiquitination
104
what are the signaling endosomes created by B arrestin good for?
they spatially and temporally extend GPCR signaling - prolongation of GPCR signaling
105
GPCR ubiquitination does what?
targeted for degradation by proteasome
106
what is the rationale of the paper?
study the intracellular trafficking of a GPCR in this case sst2A somatostatin receptor in vivo following agonist stimulation
107
what is OCT?
sst2r agonist
108
what happens to the sst2Arlocalization in the hippocampus after OCT stimulation?
moves from dendrites (molecular layer) to the cell bodies (dentate gyrus and stratum pyramidal)
109
where is sst2 in the dentate gyrus before stimulation?
in the molecular layer
110
in figure 2, the dark equals what?
lots of fluorescence aka lots of sst2A
111
how long does it take for sst2a to fully come back to the molecular layer after OCT stimulation?
7 days
112
why do they care about somatostatin?
it has inhibitory activity that could be useful for many condition like strokes or epilepsy comes from damage from excitotoxicity
113
why did measure sst2 mRNA levels? what did they find?
to see if sst2a was going to the cell body for neosynthesis.
no change in mRNA = does not go to nucleus for neosynthesis
114
if sst2a doesn't go to the cell body for neosynthesis, why does it?
for sequestration and RECYCLING
115
what is CHX?
drug that inhibits ribosome
116
what happened to sst2a after cells treated with CHX? what can we conclude?
does not affect sst2a internalization.
again shows that sst2a internalization is not for neosynthesis
117
what did they find using immunogold immunocytochemical staining of sst2?
that it goes to the golgi after OCT stimulation
118
why did they use confocal microscopy?
to prove sst2A goes to TGN using sst2 and golgi/ER markers
119
what markers did they use for ER and cis-golgi?
PDI: marker for the endoplasmic reticulum; GM130: cis-Golgi marker
120
did they find colocalization with ER or cis-golgi? (PDI / GM130)
no
121
what marker did they use for TGN?
syntaxin 6
122
confocal microscopy showed that recycling is over (back to normal) after how long?
48h
123
why are the findings not so accurate anymore?
syntaxin 6 is not a golgi marker!
124
what did adding colchicine do? conclusion?
toxin depolarizes microtubules -> stopped sst2 internalization.
sst2 transport to TGN is microtubule dependent.
125
what is the significance of the study?
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!
