G-protein coupled receptors (lect 18-20) Flashcards
what type of G proteins are Gi/o proteins?
inactivating g proteins that inactivate AC
what senses are dependent on GPCRs?
sight, taste, smell
give examples of signal molecules
proteins, small peptides, derivatives of aa, faaty acids, photons, molecules for smell and taste
give an example of a GPCR involved in asthma and the type of drug it can be used by?
b2-adrenergic receptor agonists for asthma
give an example of a GPCR involved in hypertension and the type of drug it can be used by?
b-adrenergic receptor antagonists for hypertension
give an possible experimental setup to solve the challenge ivolving GPCR structure?
(Challenge = it is TM) solubilize membrane with a detergent
what are GPCR’s most variable structures?
N and C terminus, and the extra/intracellular loops (basically the non TM domains)
where is the N and the C terminus located?
N-terminus is EXTRACELLULAR
C-terminus is INTRACELLULAR
what is a protein disulfide isomerase?
enzyme that helps generate the disulfide bond in GPCR (usually hardly found in the cell)
the disulfide bonds are created between what residues?
cysteines
GPCR cysteine must be activated by what?
palmitoylation
where on the GPCR does palmitoylation happen?
C terminal
is palmitoylation reversible?
yes
is disulfide bonding reversible?
no
what the inhibitory neurotransmitter of the CNS!!
GABA
name the GPCR families
rhodopsin, secretin receptor, glutamate receptor, adhesion, frizzled/TAS2
is there sequence similarity between families of GPCRs?
no
usually, ligand bind what part of GPCR? what is the exception?
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
are ligands specific to GPCRs?
no (One ligand can bind to different GPCRs; One GPCR can bind to different ligands)
the same effectors can cause different consequences in different what?
tissue
what is the precursor for serotonin?
L-tryptophan
what modifications happen to L-tryptophan for it to become serotonin?
hydroxylation and decarboxylation
where is 5-HT (serotonin) produced?
gut and CNS
name some of its effect
gut movements, regulation of mood, appetite, sleep, muscle contraction
how many sub-familie sof 5-HT are there? what about types of different receptors?
7 sub-families; 14 different 5-HT receptors
what is special about CCK1 cholecystokinin receptor?
it is specific to SULFATED CCK
what is CCK2 specififc for?
sulfated CCK, non-sulfated CCK, gastrin
what is luciferase? (Rluc)
enzyme that converts lenterazine into a bioluminescent compound; used for BRET
how does bret work briefly?
- 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
name 3 different ways of how GPCR dimerizeand what receptors use each method
- disulphide bond formation: calcium and glutamate R
- coiled-coil interaction via C-terminus: GABAb R
- transmembrane interactions: B2-adrenergic, dopamine
how does transmembrane interactions between GPCRs work?
they “mix”/exchange 2 of their TM helices
via what type of interactions do GPCR do transmembrane interactions?
hydrophobic interactions
what are the proposed roles of dimerization / oligomerization of GPRCs?
protein folding
efficient signal transduction
G-protein selectivity
signal modulation
what’s special about GRB1 and GRB2 GPCRs?
they need to form a dimer together to be able to signal
why can’t GBR1 signal alone? what about GBR2?
- GBR1 has a signal retention signal that keeps it in the ER
- GBR2 can get to the membrane but can not signal
what happens when GBR1 and 2 dimerize?
the dimarization hides GBR1 ER retention signal
what is Kd?
dissociation constant: ligand concentration where 50% is bound to the receptor and 50% is free
what is the formula for Kd?
Kd = [A] x [B] / [AB]
what is a saturation isotherm radioligand binding study?
experiment that measures the specific binding of the radioligand at equilibrium across a range of radioligand concentrations
what can be the axis of a plot showing saturation isotherms?
y = bound ligand; x = free ligand
or
y = bound/free ligand; x = bound ligand
what does Kd represent?
the ligand’s affinity to the receptor
what does the displacement analysis radioligand binding studies allow to determine?
the affinity of the GPCR for different agonists and antagonists
what is on the axis of the displacement analysis graph?
x = peptide conc (in the example, its in log [peptide] (M))
y = % specific binding
what is the principle behind a displacement analysis?
The unlabeled competing ligand “displaces” the radioligand from the receptor binding sites, causing a decrease in the specific binding of the radioligand.
give an idea of how you can de-orphan GPCRs
knock-in/knock-out the receptor in cell culture to get an idea of the function and hypothesize ligands
what are challengers when it comes to de-orphanizing GPCRs?
- 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
what domain is conserved in all members of the G protein superfamily?
GTPase domain
all Galpha subunits (except Galpha-i) of G proteins are postranslationally modified with what fatty acid? at what end?
plamitate at the N-terminus
what is the posttranslational modification of Galpha-i protein?
myrostoylated at the N-terminus
what are the major families of trimeric G-proteins and their role?
Gas = stimulate AC
Gai/o = inhibit AC
Gaq,11 = stimulates phospholipase CB
Ga12,13 = stimulates some GEFs
GBy = K+ channels and AC isoforms
what posttranslational modification happens to Gy subunit of G protein?
isoprenylation (geranylgeranyl, farnesyl)
where are g proteins usually located?
cytoplasmic face of the plasma membrane
how are g proteins activated?
activated GPCR acts like a GEF -> G protein alpha subunits becomes GTP bound
what happens once the G protein is activated?
conformational change exposes subunit surfaces allowing the interaction with the target
what are RGS?
regulator of G-protein signaling (acts as a GAP to shut off the signal)
Gbeta and Gy form a what? what can dissociate it?
functional unit via their coiled-coiled N terminus that can only be dissociated under denaturing conditions
what are the 2 models of G-protein activation? which one is favored right now?
collision coupling or precoupling (G protein is already coupled to the receptor).
precoupling model is favored
what happens to the G protein once it gets activated?
is dissociates from the GPCR
how is cAMP synthesized?
synthesized by AC from ATP
what continuously destroys cAMP? it becomes what?
AMP phosphodiesterase destroys cAMP into AMP
describe AC
adenylyl cyclase: large multipass membrane protein; has catalytic activity on the cytosolic side
how do Gi G proteins inhibit AC?
mainly by regulating ion channels
what are the effect of Gs proteins on cAMP?
they stimulate AC which increases cAMP
what are the targets of the cholera and the pertussis toxin?
cholera toxin targets Galpha-s G proteins.
pertussis targets Galpha-i G proteins
what are the effects of the cholera toxin? (vibrio cholerae)
it catalyzes the ribosylation of Gas subunit, rendering it constituvely active (can’t hydrolyze GTP).
causes efflux of Cl- and water into gut -> diarrhea
what does the pertussis toxin do?
catalyzes ADP ribosylation of the Galpha-1 subunit which prevents the protein from interacting with receptors by keeping it in GDP-bound form
what are cholera and pertussis toxins used for in research?
to determine whether a cells’s GPCR-dependent response is mediated by Gs or Gi
what enzyme does the ADP-ribosylation of g proteins?
Mono-ADP-ribosyltransferase transfers an ADP-ribose residue
where is the ADP-ribose added on the G protein?
- arginine for Galpha-s and beta subunit
- cysteine for Galphai/o subunit
what gets released when the G protein is ribosylated?
nicotinamide
where does the ADP-ribose residue added to the g protein come from?
from betaNAD+
what enzyme removes ADP-ribose from the g protein and how?
Specific ADP-ribosylhydrolases hydrolyze the aminoacid-ADP-ribose glycosidic linkage, thus regenerating free arginine or cysteine and releasing ADP-ribose
what protein mediates most of cAMP’s effects?
PKA
how does PKA exert its function?
cAMP binds rits regulatory subunit, catalytic subunits dissociate and phosphorylate serine and threonine residues in target proteins
what enzyme keeps cAMP low in unstimulated cells?
phosphodiesterase
explain the inhibitory loops that allows PKA to have strong brief pulses of activation
PKA activated -> activates phosphodiesterase -> lowers cAMP -> stops PKA activity
what residues are phosphorylated by PKA? is that specific?
arginine (L) - Serine (S)
or
Threonine (T) - X
(not specific)
what are AKAPs?
regulatory proteins that helps localize kinases like PKA
what can AKAPs bind?
GPCRs, substrates, kinases, cytoskeleton, membranes, and other enzymes (ex phosphodiesterase and phosphatases)
why are AKAPs important?
because PKA is not specific (it phosphorylates every L-S / T-X sequence)
give the cascade of event triggered by GPCR activation that leads to the fast effect of cAMP
- GPCR activation
- G protein activation
- Galpha-s activates AC
- production of cAMP
- activates PKA
- phosphorylate target protein
give the example of the slow effect of cAMP seen in class (cascade of events)
(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
give 2 examples of other direct ways of action of cAMP
- 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
appart from AC, what other protein is often activated by g proteins?
PLC-B (phospholipase C beta)
what type of G proteins activate PLC-B?
Gq proteins
what is the effect of PLC-B? give the cascade of events
- it cleaves PIP2 in the membrane to give IP3 and diacylglycerol
- IP3 activates IP3-gated Ca++ channels
- Ca++ increases in the cytosol
- Ca++ with diacyglycerol activate conventional PKC
- phosphorylate target proteins
what roles can diacylglycerol have?
- activate conventional PKCs (with Ca++)
- gets cleaved to produce arachninoid acid and then prostaglandins
what are the effects of prostaglandins?
inflammation, vasodilation
explain the strategies used to map G protein coupling sites
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
why did they swap intracellular loops in the experiment?
the intracellular loops of GCPRs are key regions for G protein coupling
name effects of cAMP (PKA activation) and the tissue/hormone they are involved with
- muscle: adrenaline -> glycogen breakdown
- heart: adrenaline -> increase heart rate and contractions
- ovary: LH -> progesterone secretion
- thyroid gland -> TSH -> thyroid hormone synthesis and secretion
name effects of IP3/DAG (PKC) and the tissue/hormone they are involved with
- liver: vasopressin -> glycogen breakdown
- pancreas: acetylcholine -> amylase secretion
- blood platelets: thrombin -> aggregation
what is the major reason for drug-tolerance, tachyphylaxis, and drug addiction?
down-modulation of GPCRs (desensitization)
what are the 3 levels of turning off the GPCR signal?
- deactivation: extinction of the input signal
- regulation: adjustments in the signaling apparatus
- desensitization
what are the 2 methods for the extinction of chemical messengers?
dilution (ex re-uptake of neurotransmitters) or metabolic inactivation
how is regulation of GPCR signal done? (2nd step)
- phosphodiesterase (decrease cAMP)
- phosphatases (dephosphorylate PKA targets)
- G i/o proteins inhibit
- RGS (regulator of G protein signaling)
how is desensitization of GPCR done?
- via GRK or B-arrestion (inhibibt G protein interaction with GPCR)
- receptor downmodulation
what is the main mechanism of GPCR regulation?
GRKs / B-arrestin
what domains are present in GRK structure?
- GPCR binding domain (n-term)
- RGS domain (regulator of G protein signaling)
- plasma membrane targetting domain that gets post-translationally modified (c-term)
how do GRKs get activated?
Activated form of the GPCR receptor allosterically activates the GRKs
how do GRKs and B-arrestin stop GPCR signal?
GRK phosphorylates GPCR, making B-arrestion bind the receptor and inhibiting its binding to signaling G protein
appart from an activated GPCR, what else can activate GRKs?
PKA and PKC
what happens to the GPCRs once B-arrestin binds?
B-arrestin work as an adaptor protein for clathrin and the complex gets endocytoses and recycled or degraded
what players are involved in clathrin endocytosis?
- AP2
- Arf6
- ARNO (Arf6 GEF)
- clathrin
- large GTPase dynamin (for pinching off)
what does B-arrestin regulate?
the faith of the GPCR: rapid or slow recycling, or degradation
how do GPCRs get recycled?
pH change in endosomes causes the GPCR to become a target for phosphatases -> gets dephosphorylated
what causes GPCR to get degraded?
B-arrestin interacts directly wiht ub ligase
what happens to GPCR localization when stimulated with its ligand?
moves from the membrane to the inside of the cell
