Week 11: Cell signaling pt 2 Flashcards
G-protein coupled receptors
7 transmembrane alpha helices with a C terminal that sticks out
- communicate info from the outside to the inside of the cell and are a major drug target for small molecule therapies
- they have a small pocket toward the outside of the receptor side where the ligand will bind
GPCR steps (5)
- ligand binding ⇒ anything that binds and activates
- Change in GPCR conformation
- Interaction with G protein heterotrimer
- GDP exchange for GTP (GEF)
- Activation of target proteins
heterotrimer
composed of an alpha, beta, and gamma subunits ⇒ refer to the G protein
- Binds with GDP switching to GTP in the G protein when active
- uses GEF to switch out GDP to GTP
- the alpha stays by itself and beta and gamma always stay together
common small molecules from active GPCR pathways
cAMP, IPX, K+, Ca2+
5 key facts of GPCR’s
- stimulation of GPCRs activates heterotrimeric G proteins
- some G proteins directly regulate ion channels
- some G proteins activate membrane bound enzymes
- cyclic AMP pathway can activate enzymes and turn on genes
- Inositol phospholipid pathway triggers a rise in cytosolic CA2+
heterotrimeric G proteins
(large G proteins) covalently bound to membrane lipids by their alpha and gamma subunits
Alpha subunit with bound GDP is inactive
What is like G proteins
Ras is similar to G proteins but are smaller
what attaches the G proteins to the cell membrane?
lipid modifications on both the alpha and beta/gamma portions
- when bound to GDP it is inactive
activation process of G proteins (4)
- Signal binds GPCR
- GDP to GTP exchange via GEF
- Release of G protein in active form
- Both alpha subunit and beta/gamma complex can interact wtih downstream targets
- Activates G-proteins can be stimulatory (Gs) or inhibitory (Gi) ⇒ Gq is neither inhibitory or stimulatory
where does the GDP molecule binds in G proteins?
in the AH domain of the alpha subunit
what does the active G protein do?
- Activated alpha subunit (with GTP) activates or inhibits the target and stays active until it is hydrolyzed ⇒ time delay from active to hydrolysis and during this window it keeps activating the effect
- Active beta/gamma can also activate or inhibit the target
how do turn off the G protein
GTP hydrolysis terminates (via GAP) activation of G protein
- Inactivated alpha GDP subunits dissociate from target and reforms inactive G protein complex
cholera toxin (CTx)
binds to the G protein and modifies alpha subunit of stimulatory G proteins so they can no longer hydrolyze bound GTP ⇒ signals cannot be turned off
what does cholera toxin shut off?
Shuts off the GAP activity which prevents hydrolysis leading to cAMP which impacts CFTR regulating Cl- kept in the cell
what is the effect of cholera toxin?
Causes excessive outflow of Cl- and H2O into gut, leading to diarrhea and dehydration
- As Cl- goes out so does sodium in the cell junctions with water ⇒ lose sodium and water from the system
how is cholera toxin treated?
you treat cholera with antibiotics but also electrolytes filled with NaCl
what secondary molecules are influenced by cholera toxin?
works in GalphaS to keep it active in GTP state which activates adenylyl cyclase to increase cAMP
pertussis toxin (Ptx)
modified alpha subunit of inhibitory G-proteins and locks them into inactive GDP bound state
- Inhibiting an inhibitory G-protein results in prolonged signaling that stimulates coughing (whooping cough)
what does pertussis toxin keep inactive?
it works on Galpha to keep it inactive in the GDP bound state
what effect does pertussis toxin have on second messengers?
it inhibits adenylyl cyclase which increases cAMP because it inhibits adenylyl cyclases inhibitor
which 4 G proteins decide downstream pathways?
- Gbeta-gamma
- GalphaS
- GalphaQ
- GalphaT aka transducin
Gbeta-gamma
activates ion channels
GalphaS
activates adenylyl cyclase which makes cAMP
GalphaQ
activates inositol phosphate which activates calcium signaling
GalphaT
affects photoreceptor signaling
process of how Gbeta-gamma regulates potassium channels? (5) What’s the result?
- Ligand is acetylcholine (Ach) binds GPCR
- Activates G-protein
- Active beta/gamma complex to stimulate opening of K+ ion channel ⇒ K+ goes out
- GTP hydrolysis inactivates G-protein
- K+ channel closes
→ Results in relaxation of the muscle cells
which G protein activates membrane bound enzymes?
GalphaS G protein gets activated by GPCR and then activates adenylyl cyclase (attached to it) which takes ATP and converts it to cAMP
were GPCRs or G proteins found first by Nobel prize winners?
G proteins were first by Gilman after Rodbell discovered cAMP
- GPCRs were discovered later in 2012
adenylyl cyclase
converts ATP to cyclic AMP, cAMP activates targets
- Take out 2 phosphate and grab the remaining phosphate to be stitched toward the back of the molecule ⇒ 1 phosphate remains
- Starts the signal and amplifies the signal
cAMP phosphodiesterase
converts cAMP to AMP to terminate a signal
- Some G-proteins stimulate adenylyl cyclase, others inhibit
what does caffeine inhibit?
phosphodiesterase which prolongs cAMP signaling in the nervous system
- cAMP has many important targets
how does cAMP signaling in skeletal muscles work?
- Adrenaline activates the GPCR protein which activates GalphaS connected to adenylyl cyclase
- GalphaS Stimulates adenylyl cyclase to make cAMP
cAMP activates PKA - PKA activates a phosphorylase kinase which causes a phosphorylation cascade
- Stimulates glycogen breakdown to glucose for energy in muscle via glycogen phosphorylase
is adrenaline fast or slow acting?
fast
adrenaline
fight or flight and binds to a GPCR
how does cAMP work to turn on genes?
- same original pathway as adrenaline activated by a signaling molecule and then activates GalphaS and adenylyl cyclase
- adenylyl cyclase makes cAMP which binds PKA
- Active PKA goes to nucleus
- PKA activates CREB via phosphorylation
- CREB recruits a CREB binding protein (CBP) which binds to the cAMP response element (CRE)
- this binding induces gene transcription of the target gene
what does Pi transcription factor (CREB) do?
stimulates transcription and effects depend on cell types and specific transcription factors affected ⇒ is the transcription factor
- Convert short term signals into long term response
what does the slow cAMP/PKA pathway result in? (2)
- Secretion of hormones in endocrine system
- Learning and memory in brain ⇒ encode changes to remember what the event was
what are downstream of GaphaS? (4)
- Activation of adenylyl cyclase to produce cAMP (fast)
- Activation of PKA to phosphorylate CREB (slow)
- Activation of PKA to phosphorylate phosphorylase kinase (fast)
- Translocation of PKA to the nucleus (slow)
what is the inositol phospholipid pathway? (5)
- a ligand binds GPCR
- this activates GalphaQ
- this activates phospholipase C-beta to cleave IP3 from PI(4,5)P2 lipid in the membrane (results in diacylglycerol in the membrane still)
- the cleaves IP3 stimulates calcium channels to open in the ER membrane and to activate protein kinase C binding to diacylglycerol
- calcium floods out of the ER lumen and binds to protein kinase C attached to diacylglycerol on the plasma membrane
PLC beta
lipase that cleaves a lipid by cutting off the IP3 unit off of PI(4,5)P2 leaving the diacylglycerol portion in the plasma membrane
what is protein kinase A downstream of vs protein kinase C?
protein kinase A is downstream of cAMP and protein kinase C is downstream of IP3 and Ca2+
diacylglycerol
binds a kinase called protein kinase C
second messenger
any intermediate that is amplifying the signal
how to turn off the G protein signal?
when activated, the GPCR can be phosphorylated by a GPCR kinase (GRK) which attracts arresting to block the signal from continuing
receptor tyrosine kinases (RTKs)
comes from the receptor itself being a kinase
- There are 2 receptors on the membrane which are separate from one another
- the knob is the tyrosine kinase domain
how do RTK’s work?
Signal growth factor (protein) binds and then dimerizes the receptor to bring the two together
- The tyrosine kinase trans-autophosphorylation one another leading to the receptor being active ⇒ the kinase is not active so it can phosphorylate other sites for binding
- Phosphorylated sites allow for scaffolding of other adaptor proteins which bring something else onto the scaffold
- end goal is to relay the signal downstream leading to what the receptor is propagating
what do growth factors signal through?
RTKs => most have a growth factor in the name because activation of the receptor stimulates growth and division
- Often increases protein synthesis, proliferation (cell division), growth, and survival
RTKs have structural diversity T/F?
True
1. Comes from having diverse extracellular domains ⇒ sizes and types
2. All share the tyrosine kinase domain on the inside of the cell membrane
EGFR RTK activation process?
- EGF is the ligand that binds to activate the RTK
- There is a receiver kinase and an activator kinase
- Once the C terminal tail is phosphorylated, these will scaffold other proteins
there are different phosphorylated tyrosine sites that go on to do what?
recruit different downstream proteins
Ras
a monomeric G-protein anchored to the membrane
- Ras-GTP activates many downstream targets and mutations in Ras account for a vast majority of cancer
steps for activating Ras
- Activated RTK
- Grb2 adaptor recruits another adaptor Sos (2 different proteins)
- There are SH3 proteins which are universal adaptors between two proteins
- Sos has a Ras GEF domain (GEF for Ras as the inactive G protein) which is different from the larger G proteins
- Has GDP sitting on the membrane and is activated from GDP getting exchanged for GTP
a signaling protein only made up of one SH2 domain and 2 SH3 domains is likely what?
an adaptor protein => SH2 and SH3 domains are a hallmark of adaptors
what is Ras known to activate?
MAPK => mitogen activated protein kinase
mitogens
something that causes mitosis
- There must be something that phosphorylates the MAP kinase
Ras activates ___ which activates ____
Ras activates Mek which activates Erk
- Blockbuster drugs target Ras when you get a melanoma
ERK1/2
a key stimulator of cell growth and proliferation
- the end goal is that you activate other proteins and trigger gene expression
how does activation of PIP3 signaling happen?
- RTK activates PI 3-kinase and Pi 3-kinase converts membrane bound PIP2 to PIP3 (lipid) => If you cut off the phosphate you get diacylglycerol and if you add a phosphate you get PIP
- PIP3 acts as a second messenger and recruits protein kinase 1 and Akt kinase (PKB) which scaffold mTOR
- Akt kinase promotes cell survival and cell growth by splitting the Bcl2 and Bad
Akt kinase phosphorylates which protein? This does what?
Bad; this releases Bcl2 and binds 14-3-3 adaptor protein so that active Bcl2 inhibits apoptosis
what is the mTOR pathway?
- activated RTK activates PI 3-kinase to activate AKT
- AKT inhibits TSC which is Rheb’s GAP protein
- TSC cannot hydrolyze Rheb to turn off the signal so Rheb can now activate mTOR which stimulates cell growth
alternative mTOR pathway?
- activated RTK activates Ras which activates Erk (MAPK)
- ERK inhibits TSC which is Rheb’s GAP protein
- TSC cannot hydrolyze Rheb to turn off the signal so Rheb can now activate mTOR which stimulates cell growth
Rheb
when this is bound to GTP it activates the mTOR complex which stimulates growth
Growth
refers to a cell getting bigger
proliferation
refers to the cell multiplying
Rapamycin
has an ability to inhibit mTOR and can be used to suppress the immune system
mTOr in complex 1 contains the protein raptor which is sensitive to rapamycin and stimulates cell growth T/F?
True
rapamycin (sirolimus) is used as an immunosuppressant for preventing organ transplant rejection T/F?
True
Akt activates mTOR in complex by activating a Rheb-Gab called Tsc2 T/F?
False
mTOR in complex 1 is activated in the presence of growth factors T/F?
True
