Final: Ch 15 G-Protein Coupled Receptors Flashcards
what is a G-protein
GTP-binding protein
bind GTP/GDP
active when bound to GTP, inactive when bound to GDP
regulation of G-protein genes
transcriptional regulation using TF (binds to promoter)
post-translational regulation/modification
examples of post-transcriptional regulation of G-protein genes
alternative splicing
capping
polyadenylation
RNAi
G-proteins are anchored in the cell membrane when…
GDP bound –> ligand binds –> GTP binds to alpha subunit
4 things GPCR signal transduction pathways have in common
receptor with 7 transmembrane alpha helices
coupled trimeric G-protein switch
membrane-bound effector protein
proteins for feedback regulation and desensitization of the pathway
where are the N and C termini of GPCR
N-terminus on extracellular face
C-terminus on cytosolic face
B-adrenergic receptors
bind hormones like epi and nor-epi
can different subtypes of GPCR bind the same hormone and have different effects?
yes
ex. epinepherine (in heart increases contraction, in smooth muscle relaxes)
what happens in fight or flight
adrenal gland releases Epi to bind B-adrenergic GPCR in liver/muscle cells
stimulate glycogen breakdown to glucose
high epi in blood
trimeric G-proteins 3 subunits
alpha
beta + gamma (complexed together)
linked to membrane by lipids
in resting state, G-alpha subunit has a bound ___ and is complexed to _____
GDP, complexed to G-betagamma
binding of ligand to GPCR changes conformation of the receptor and allows…
the receptor to bind to G-alpha subunit –> release GDP
GTP binds G-alpha –> release of receptor and G-betagamma
G-alpha binds effector protein
how to detect GPCR-mediated dissociation of the G protein
FRET - fluorescence energy transfer
all effector proteins in GPCR pathway are either…
membrane bound ion channels
membrane bound enzymes that catalyze formation of 2nd messengers
which subunit of the G-protein determines its function?
G-alpha
ex. G-alpha S activates adenylyl cyclase to increase cAMP
G-alpha i inhibits adenylyl cyclase to decrease cAMP
one of the simplest cellular responses to a signal is the opening of…
ion channels
ex. open ligand-gated ion channels for neurotransmitters
many nt receptors are…
GPCR whose effector proteins are Na or K channels
mAChR are a type of GPCR found in _____ muscle
cardiac muscle
what happens when mAChR are activated
G-betagamma opens K+ channel (usually G-alpha)
slow heart rate when K+ channels open
efflux of K+ causes hyperpolarization and slows contraction
cones are for ____, rods are for ____/______
color, black/white
found in retina, signals processed by visual cortex
rhodopsin
light-sensitive GPCR (opsin) that helps rods sense light
G-protein called Transducin (G-alpha-t)
photon-absorbing pigment called retinal
how does rhodopsin differ from other GPCRs
absorption of a photon of light is the signal (by retinal), not binding of ligand
what happens when rhodopsin absorbs a photon of light
cis –> trans in retinal form causes conformational change in GPCR opsin
rhodopsin binds G-alpha-t subunit and exchanges GDP for GTP
G-alpha-t removes inhibitory gamma segments from phosphodiesterase (PDE), which turns cGMP –> GMP
activation of rhodopsin by light leads to the closing of ____-gated cation channels
cGMP
causes membrane potential to become more inside negative (hyperpolarization-less nt released)
in the dark the membrane potential of a rod cell is…
-30mV
this is a state of depolarization so in the dark, the rod cell is constantly secreting nt
why is there a constant state of depolarization of resting rod cells
large number of open nonselective ion channels that let Na and Ca in
to close the cation channels, the 2nd messenger _____ is turned into
cGMP –> GMP
upon absorbing light, cGMP PDE hydrolyzes cGMP to GMP
cGMP releases from gated channels, closing them
the ____ level of cGMP in the dark keeps cGMP gated cation channels open, in the light…
high
decrease in cGMP concentration by PDE causes closing of channels and less nt release
what makes the rhodopsin signal transduction pathway so sensitive?
signal amplification of the photon of light
each activated opsin activates 500 G-alpha-t molecules which activate the PDE
what GAP proteins inactivate G-alpha-t GTP
RGS-9 and G-beta-5
hydrolyze GTP –> GDP so PDE is inactivated again
Rhodopsin kinase
phosphorylates active rhodopsin
arrestin binds after 3 phosphates and prevents transducin activation (G-alpha-t)
how do rod cells adapt to varying levels of ambient light – visual adaptation
adjusting trafficking of arrestin and transducin
80% of G-alpha-t and G-betagamma move out of the outer segment and 80% of arrestin moves into outer segment in 10 min of moderate light
prevents PDE activation
adenylyl cyclase synthesizes ____ from ____
cAMP, ATP
cAMP activates a _____
kinase
glycogen
a storage polymer of glucose
broken down in liver/muscle cells in response to the hormone epinerpherine
all of the effects of cAMP are mediated through activation of which kinase
protein kinase A (cAMP dependent protein kinase)
binding of cAMP opens catalytic active site
degradation of glycogen is called
glycogenolysis
stepwise removal of glucose residues from one end of the polymer by glycogen phosphorylase
in the liver, glycogen stores are broken down to ….
glucose, which is released into the blood and transported to tissues like muscle and brain for energy
2 ways epinerpherine stimulated activation of adenylyl cyclase –> higher cAMP –> PKA activation enhances conversion of glycogen to glucose
inhibit glycogen synthesis enzymes
stimulating glycogen degradation enzymes
what does PKA do
phosphorylates and inactivates glycogen synthase (GS), the enzyme that synthesizes glycogen
phosphorylates the inhibitor of phosphoprotein phosphatase (IP) to inactivate it (so glycogen phosphorylase kinase can break down glycogen)
in adipose cells, what does activation of PKA do
phosphorylation of a lipase that hydrolyzes stored triglycerides to FA and glycerol for energy
is there signal amplification in the cAMP-PKA pathway?
yes
GPC hormone receptors are able to diffuse rapidly in the plasma membrane
a single epinerpherine-GPCR complex activates up to 100 G-alpha s molecules –> adenylyl cyclase and so on
activation of PKA also stimulates the expression of…
many genes, including CREB
leads to long-term effects
CRE
cAMP response element
binds the phosphorylated form of the TF CREB
all genes regulated by PKA have ____
CRE
binds the phosphorylated form of the TF CREB
CREB TF
CRE-binding protein
found in nucleus
genomic response of PKA
catalytic subunit of PKA targets CREB in nucleus and phosphorylates it
CREB binds to distal promoter (response element) of target gene and recruits co-regulator
stimulates gene transcription
a ligand (hormone) can have a non ______ response, a ______ response, or both
genomic, genomic
what is A kinase-associated protein
an anchoring protein that binds PKA to specific locations so it is activated in only those locations
ex. heart muscle cells have AKAP15 near Ca channels
AKAP in the outer nuclear membrane
localizes PKA and cAMP PDE to outer nuclear membrane
helps catalytic subunits of PKA enter nucleus
allows for negative feedback regulation
3 ways to down-regulate signaling through the GPCR/cAMP/PKA pathway
affinity of receptor for ligand is decreased when GDP is replaced by GTP in G-alpha
intrinsic GTPase activity of G-alpha hydrolyzes GTP –> GDP (hydrolysis rate increased when G-alpha binds adenylyl cyclase)
cAMP PDE hydrolyzes cAMP –> AMP to end cellular response
most GPCRs are also down-regulated by ______-_________
feedback repression
end product blocks early step in pathway
B-arrestin
assists endocytosis of cell-surface receptors
phospholipase C (PLC)
enzymes that hydrolyze a phosphoester bond to yield 2nd messengers that raise cytosolic Ca2+ levels and remodel actin
activate protein kinase C
protein kinase C (PKC)
affect cellular growth and differentiation
important 2nd messengers are derived from the membrane lipid, ______________
phosphatidylinositol (PI)
PI –> PIP2
PLC cleaves PIP2 into…
DAG (diacylglycerol) and IP3 (inositol triphosphate)
DAG is associated with the ______ while IP3 freely _______ in the cytosol
membrane, diffuses
GPCR that activate PLC increase cytosolic ____ concentration even when the ions are absent from the extracellular fluid
Ca2+ concentration
PLC cleaves PIP2 into IP3, which opens IP3 gated-Ca channel in ER lumen
what does DAG activate
protein kinase C - moves to cytosolic leaflet
activation of PKC has what effects
growth and metabolism
phosphorylates TF
ex. in liver cells, PKC regulates glycogen metabolism
calmodulin
small cytosolic switch protein that mediates many cellular effects of Ca ions
glycogen breakdown and synthesis is coordinately regulated by which two second messengers
Ca2+ and cAMP
levels are regulated by neural and hormonal stimulation
why is nitroglycerin used to treat angina
decomposes to nitric oxide (NO), which relaxes smc in the heart
what is the allosteric effect (Hb) – positive cooperativity
Hb binds oxygen – increased affinity in lungs, less in tissue
more binding increases affinity
creates a more dramatic switch
basic epi GPCR pathway
epi + GPCR –> GTP G-alpha
activate adenylyl cyclase –> higher cAMP
cAMP activates PKA to stimulate breakdown of glycogen OR phosphorylation of TF in the nucleus (ex. CREB)
do G proteins usually have short-term or long-term effects?
short-term
modify existing enzymes or ion channels
structure of inactive PKA
tetramer of 2 regulatory subunits and 2 catalytic subunits
each R subunit binds the active site of a C subunit to inactivate
how is inactive PKA turned on
2 cAMP binds each R subunit
conformational change causes R subunits to release C subunits
glycogen phosphorylase kinase
glycogen –> glucose activates glycogen phosphorylase
activated by high [Ca2+], PKA
glycogen synthase
stimulates glycogen formation
inhibited by PKA, PKC
