G-protein coupled receptors (lect 18-20) Flashcards

1
Q

what type of G proteins are Gi/o proteins?

A

inactivating g proteins that inactivate AC

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2
Q

what senses are dependent on GPCRs?

A

sight, taste, smell

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3
Q

give examples of signal molecules

A

proteins, small peptides, derivatives of aa, faaty acids, photons, molecules for smell and taste

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4
Q

give an example of a GPCR involved in asthma and the type of drug it can be used by?

A

b2-adrenergic receptor agonists for asthma

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5
Q

give an example of a GPCR involved in hypertension and the type of drug it can be used by?

A

b-adrenergic receptor antagonists for hypertension

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6
Q

give an possible experimental setup to solve the challenge ivolving GPCR structure?

A

(Challenge = it is TM) solubilize membrane with a detergent

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7
Q

what are GPCR’s most variable structures?

A

N and C terminus, and the extra/intracellular loops (basically the non TM domains)

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8
Q

where is the N and the C terminus located?

A

N-terminus is EXTRACELLULAR
C-terminus is INTRACELLULAR

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9
Q

what is a protein disulfide isomerase?

A

enzyme that helps generate the disulfide bond in GPCR (usually hardly found in the cell)

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10
Q

the disulfide bonds are created between what residues?

A

cysteines

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11
Q

GPCR cysteine must be activated by what?

A

palmitoylation

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12
Q

where on the GPCR does palmitoylation happen?

A

C terminal

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13
Q

is palmitoylation reversible?

A

yes

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14
Q

is disulfide bonding reversible?

A

no

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15
Q

what the inhibitory neurotransmitter of the CNS!!

A

GABA

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16
Q

name the GPCR families

A

rhodopsin, secretin receptor, glutamate receptor, adhesion, frizzled/TAS2

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17
Q

is there sequence similarity between families of GPCRs?

A

no

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18
Q

usually, ligand bind what part of GPCR? what is the exception?

A

N terminus (because its the extracellular one DUH)
exception is rhodopsin family GPCRs:
- alpha group bind ligans in the TM domains
- beta and y group bind ligans via extracellular loop

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19
Q

are ligands specific to GPCRs?

A

no (One ligand can bind to different GPCRs; One GPCR can bind to different ligands)

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20
Q

the same effectors can cause different consequences in different what?

A

tissue

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21
Q

what is the precursor for serotonin?

A

L-tryptophan

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22
Q

what modifications happen to L-tryptophan for it to become serotonin?

A

hydroxylation and decarboxylation

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23
Q

where is 5-HT (serotonin) produced?

A

gut and CNS

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24
Q

name some of its effect

A

gut movements, regulation of mood, appetite, sleep, muscle contraction

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25
Q

how many sub-familie sof 5-HT are there? what about types of different receptors?

A

7 sub-families; 14 different 5-HT receptors

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26
Q

what is special about CCK1 cholecystokinin receptor?

A

it is specific to SULFATED CCK

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27
Q

what is CCK2 specififc for?

A

sulfated CCK, non-sulfated CCK, gastrin

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28
Q

what is luciferase? (Rluc)

A

enzyme that converts lenterazine into a bioluminescent compound; used for BRET

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29
Q

how does bret work briefly?

A
  • link protein A to Rluc and protein B to GFP
  • add lenterazine
  • shine blue light
  • green light will be emitted if proteins are in close proximity and this is measured
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30
Q

name 3 different ways of how GPCR dimerizeand what receptors use each method

A
  1. disulphide bond formation: calcium and glutamate R
  2. coiled-coil interaction via C-terminus: GABAb R
  3. transmembrane interactions: B2-adrenergic, dopamine
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31
Q

how does transmembrane interactions between GPCRs work?

A

they “mix”/exchange 2 of their TM helices

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32
Q

via what type of interactions do GPCR do transmembrane interactions?

A

hydrophobic interactions

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33
Q

what are the proposed roles of dimerization / oligomerization of GPRCs?

A

protein folding
efficient signal transduction
G-protein selectivity
signal modulation

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34
Q

what’s special about GRB1 and GRB2 GPCRs?

A

they need to form a dimer together to be able to signal

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35
Q

why can’t GBR1 signal alone? what about GBR2?

A
  • GBR1 has a signal retention signal that keeps it in the ER
  • GBR2 can get to the membrane but can not signal
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36
Q

what happens when GBR1 and 2 dimerize?

A

the dimarization hides GBR1 ER retention signal

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37
Q

what is Kd?

A

dissociation constant: ligand concentration where 50% is bound to the receptor and 50% is free

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38
Q

what is the formula for Kd?

A

Kd = [A] x [B] / [AB]

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39
Q

what is a saturation isotherm radioligand binding study?

A

experiment that measures the specific binding of the radioligand at equilibrium across a range of radioligand concentrations

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40
Q

what can be the axis of a plot showing saturation isotherms?

A

y = bound ligand; x = free ligand
or
y = bound/free ligand; x = bound ligand

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41
Q

what does Kd represent?

A

the ligand’s affinity to the receptor

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42
Q

what does the displacement analysis radioligand binding studies allow to determine?

A

the affinity of the GPCR for different agonists and antagonists

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43
Q

what is on the axis of the displacement analysis graph?

A

x = peptide conc (in the example, its in log [peptide] (M))
y = % specific binding

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44
Q

what is the principle behind a displacement analysis?

A

The unlabeled competing ligand “displaces” the radioligand from the receptor binding sites, causing a decrease in the specific binding of the radioligand.

45
Q

give an idea of how you can de-orphan GPCRs

A

knock-in/knock-out the receptor in cell culture to get an idea of the function and hypothesize ligands

46
Q

what are challengers when it comes to de-orphanizing GPCRs?

A
  • GPCR can exist as oligomers
  • Accessory protein may be required
  • Some GPCR signal without G-protein
  • Concentration of transmitter may be low or only temporarily expressed
47
Q

what domain is conserved in all members of the G protein superfamily?

A

GTPase domain

48
Q

all Galpha subunits (except Galpha-i) of G proteins are postranslationally modified with what fatty acid? at what end?

A

plamitate at the N-terminus

49
Q

what is the posttranslational modification of Galpha-i protein?

A

myrostoylated at the N-terminus

50
Q

what are the major families of trimeric G-proteins and their role?

A

Gas = stimulate AC
Gai/o = inhibit AC
Gaq,11 = stimulates phospholipase CB
Ga12,13 = stimulates some GEFs
GBy = K+ channels and AC isoforms

51
Q

what posttranslational modification happens to Gy subunit of G protein?

A

isoprenylation (geranylgeranyl, farnesyl)

52
Q

where are g proteins usually located?

A

cytoplasmic face of the plasma membrane

53
Q

how are g proteins activated?

A

activated GPCR acts like a GEF -> G protein alpha subunits becomes GTP bound

54
Q

what happens once the G protein is activated?

A

conformational change exposes subunit surfaces allowing the interaction with the target

55
Q

what are RGS?

A

regulator of G-protein signaling (acts as a GAP to shut off the signal)

56
Q

Gbeta and Gy form a what? what can dissociate it?

A

functional unit via their coiled-coiled N terminus that can only be dissociated under denaturing conditions

57
Q

what are the 2 models of G-protein activation? which one is favored right now?

A

collision coupling or precoupling (G protein is already coupled to the receptor).
precoupling model is favored

58
Q

what happens to the G protein once it gets activated?

A

is dissociates from the GPCR

59
Q

how is cAMP synthesized?

A

synthesized by AC from ATP

60
Q

what continuously destroys cAMP? it becomes what?

A

AMP phosphodiesterase destroys cAMP into AMP

61
Q

describe AC

A

adenylyl cyclase: large multipass membrane protein; has catalytic activity on the cytosolic side

62
Q

how do Gi G proteins inhibit AC?

A

mainly by regulating ion channels

63
Q

what are the effect of Gs proteins on cAMP?

A

they stimulate AC which increases cAMP

64
Q

what are the targets of the cholera and the pertussis toxin?

A

cholera toxin targets Galpha-s G proteins.
pertussis targets Galpha-i G proteins

65
Q

what are the effects of the cholera toxin? (vibrio cholerae)

A

it catalyzes the ribosylation of Gas subunit, rendering it constituvely active (can’t hydrolyze GTP).
causes efflux of Cl- and water into gut -> diarrhea

66
Q

what does the pertussis toxin do?

A

catalyzes ADP ribosylation of the Galpha-1 subunit which prevents the protein from interacting with receptors by keeping it in GDP-bound form

67
Q

what are cholera and pertussis toxins used for in research?

A

to determine whether a cells’s GPCR-dependent response is mediated by Gs or Gi

68
Q

what enzyme does the ADP-ribosylation of g proteins?

A

Mono-ADP-ribosyltransferase transfers an ADP-ribose residue

69
Q

where is the ADP-ribose added on the G protein?

A
  • arginine for Galpha-s and beta subunit
  • cysteine for Galphai/o subunit
70
Q

what gets released when the G protein is ribosylated?

A

nicotinamide

71
Q

where does the ADP-ribose residue added to the g protein come from?

A

from betaNAD+

72
Q

what enzyme removes ADP-ribose from the g protein and how?

A

Specific ADP-ribosylhydrolases hydrolyze the aminoacid-ADP-ribose glycosidic linkage, thus regenerating free arginine or cysteine and releasing ADP-ribose

73
Q

what protein mediates most of cAMP’s effects?

74
Q

how does PKA exert its function?

A

cAMP binds rits regulatory subunit, catalytic subunits dissociate and phosphorylate serine and threonine residues in target proteins

75
Q

what enzyme keeps cAMP low in unstimulated cells?

A

phosphodiesterase

76
Q

explain the inhibitory loops that allows PKA to have strong brief pulses of activation

A

PKA activated -> activates phosphodiesterase -> lowers cAMP -> stops PKA activity

77
Q

what residues are phosphorylated by PKA? is that specific?

A

arginine (L) - Serine (S)
or
Threonine (T) - X
(not specific)

78
Q

what are AKAPs?

A

regulatory proteins that helps localize kinases like PKA

79
Q

what can AKAPs bind?

A

GPCRs, substrates, kinases, cytoskeleton, membranes, and other enzymes (ex phosphodiesterase and phosphatases)

80
Q

why are AKAPs important?

A

because PKA is not specific (it phosphorylates every L-S / T-X sequence)

81
Q

give the cascade of event triggered by GPCR activation that leads to the fast effect of cAMP

A
  1. GPCR activation
  2. G protein activation
  3. Galpha-s activates AC
  4. production of cAMP
  5. activates PKA
  6. phosphorylate target protein
82
Q

give the example of the slow effect of cAMP seen in class (cascade of events)

A

(GPCR activatin, G protein activation, Galpha-s activates AC, cAMP production, PKA activation)
- PKA enters nucleus and phosphorylates CREB
- phosphorylated CREB binds CBP
- complex binds CRE on DNA
- activates gene transcription of SOMATOSTATIN

83
Q

give 2 examples of other direct ways of action of cAMP

A
  • cAMP can directly activate special ion channels in the plasma membrane (ex olfactory neurons)
  • directly activates a guanine nucleotide exchange factor (GEF); activates monomeric GTPase (Rap1); increased cell adhesion through activation of integrins
84
Q

appart from AC, what other protein is often activated by g proteins?

A

PLC-B (phospholipase C beta)

85
Q

what type of G proteins activate PLC-B?

A

Gq proteins

86
Q

what is the effect of PLC-B? give the cascade of events

A
  1. it cleaves PIP2 in the membrane to give IP3 and diacylglycerol
  2. IP3 activates IP3-gated Ca++ channels
  3. Ca++ increases in the cytosol
  4. Ca++ with diacyglycerol activate conventional PKC
  5. phosphorylate target proteins
87
Q

what roles can diacylglycerol have?

A
  1. activate conventional PKCs (with Ca++)
  2. gets cleaved to produce arachninoid acid and then prostaglandins
88
Q

what are the effects of prostaglandins?

A

inflammation, vasodilation

89
Q

explain the strategies used to map G protein coupling sites

A

swap different intracellular loops and see the effect on AC vs PLC-B activation (phosphatidylinositol); this will tell you what loop is involved in which pathway

90
Q

why did they swap intracellular loops in the experiment?

A

the intracellular loops of GCPRs are key regions for G protein coupling

91
Q

name effects of cAMP (PKA activation) and the tissue/hormone they are involved with

A
  • muscle: adrenaline -> glycogen breakdown
  • heart: adrenaline -> increase heart rate and contractions
  • ovary: LH -> progesterone secretion
  • thyroid gland -> TSH -> thyroid hormone synthesis and secretion
92
Q

name effects of IP3/DAG (PKC) and the tissue/hormone they are involved with

A
  • liver: vasopressin -> glycogen breakdown
  • pancreas: acetylcholine -> amylase secretion
  • blood platelets: thrombin -> aggregation
93
Q

what is the major reason for drug-tolerance, tachyphylaxis, and drug addiction?

A

down-modulation of GPCRs (desensitization)

94
Q

what are the 3 levels of turning off the GPCR signal?

A
  1. deactivation: extinction of the input signal
  2. regulation: adjustments in the signaling apparatus
  3. desensitization
95
Q

what are the 2 methods for the extinction of chemical messengers?

A

dilution (ex re-uptake of neurotransmitters) or metabolic inactivation

96
Q

how is regulation of GPCR signal done? (2nd step)

A
  • phosphodiesterase (decrease cAMP)
  • phosphatases (dephosphorylate PKA targets)
  • G i/o proteins inhibit
  • RGS (regulator of G protein signaling)
97
Q

how is desensitization of GPCR done?

A
  • via GRK or B-arrestion (inhibibt G protein interaction with GPCR)
  • receptor downmodulation
98
Q

what is the main mechanism of GPCR regulation?

A

GRKs / B-arrestin

99
Q

what domains are present in GRK structure?

A
  • GPCR binding domain (n-term)
  • RGS domain (regulator of G protein signaling)
  • plasma membrane targetting domain that gets post-translationally modified (c-term)
100
Q

how do GRKs get activated?

A

Activated form of the GPCR receptor allosterically activates the GRKs

101
Q

how do GRKs and B-arrestin stop GPCR signal?

A

GRK phosphorylates GPCR, making B-arrestion bind the receptor and inhibiting its binding to signaling G protein

102
Q

appart from an activated GPCR, what else can activate GRKs?

A

PKA and PKC

103
Q

what happens to the GPCRs once B-arrestin binds?

A

B-arrestin work as an adaptor protein for clathrin and the complex gets endocytoses and recycled or degraded

104
Q

what players are involved in clathrin endocytosis?

A
  • AP2
  • Arf6
  • ARNO (Arf6 GEF)
  • clathrin
  • large GTPase dynamin (for pinching off)
105
Q

what does B-arrestin regulate?

A

the faith of the GPCR: rapid or slow recycling, or degradation

106
Q

how do GPCRs get recycled?

A

pH change in endosomes causes the GPCR to become a target for phosphatases -> gets dephosphorylated

107
Q

what causes GPCR to get degraded?

A

B-arrestin interacts directly wiht ub ligase

108
Q

what happens to GPCR localization when stimulated with its ligand?

A

moves from the membrane to the inside of the cell