Functions of G protein-coupled receptors in cells Flashcards
GPCRs fast acting effectors (K+ channels)
ex, muscarinic acetylcholine receptor in the heart
Fast acting receptors are usually associated with channel opening
- acetylcholine binds to its receptor in cardiac cells —> Removal of GDP and insertion of GTP. and normally the alpha subunit would move, but this is an exception where you have betagamma subunit moving along membrane going to the K+ channel (effector protein) to activate it!
- this opens the K+ channel and cellular hyperpolarisation happens which slows the rate of heart muscle contraction
Regulation of Adenylyl Cyclase
- adenylyl cyclase (E) catalyzes the formation of cAMP
- GPCR/cAMP pathway is very common in mammalian cells
- Stimulatory/Inhibitory G protein complex activates/inhibits catalyzing activity of E, depending on what receptor is expressed (receptor for stimulatory/inhibitory hormone)
The inappropriate activation of Adenylyl Cyclase: Cholera
- causes the Cholera Toxin to be produced (extracellularly) by the bacteria Cholera which is found in contaminated drinking water
- toxin enters enterocytes of small intestine using a GM1 ganglioside receptor
- toxin maintains GalphaStimulatory in active state (GTP) —> mass activation of Adenylyl Cyclase—> massive increase in cAMP levels (=secondary messenger that can bind to things)—> hyperactivity of CFTR ion channel –> Cl- pumped out and Na+ follows + H2O follows –> massively dehydrated because they’re not retaining any water in intestine —> massive diarrhea
Inappropriate activation of Adenylyl Cyclase: Whooping Cough – Pertussis Toxin
- Bordetella pertussis secretes its toxin (extracellularly) which enters ciliated epithelial cells in lungs
- pertussis toxin maintains GalphaInhibitory in inactive state (GDP) by binding to it (and so now there’s nothing to inhibit Gas)—> mass activation of Adenylyl Cyclase –> mass increase in cAMP levels –> increase activity in ion pumps (CFTR)–> movement of ions into trachea and bronchiole spaces and subsequent movement of water there too —> mucous secretion and electrolyte/H2O accumulation in lungs –> cough out watery mucus
How does cAMP activate protein kinase A?
- Protein kinase A is composed of two catalytic subunits and 2 regulatory subunits
- cAMP binds regulatory subunits in a cooperative fashion (= binding the first cAMP to CNB-B lowers the Kd to bind the second cAMP to CNB-A… this is on each regulatory subunit)
- when they bind then the regulatory subunits dissociate from the catalytic ones
What are the 4 common second messengers, and what are each of their functions?
cAMP: activates PKA
cGMP: activates PKG, and opens cation channels in rod cells
DAG: activates PKC
IP3: opens Ca2+ channels in the Endoplasmic reticulum
Signal Transduction often involves Signal Amplification. Explain.
What places can you see Fight or Flight response?
Epinephrine binds to GPCRs –> activates Adenylyl Cyclase but amplified—> produces high cAMP levels but amplified —> binds and activates PKA, which phosphorylates —> activated enzymes but amplified—>creates product –> Adrenaline rush! (glucose liberation, heart muscle contraction, vasoconstriction ~~ Fight or Flight Response – in liver, cardiac muscle, and skeletal muscle
Regulation of glycogen metabolism by increased cAMP
Think: is epinephrine present?
==> Stimulation of glycogen breakdown
○ active Protein Kinase A (indirectly from epinephrine) activates GPK (Glycogen phosphorylase kinase) by phosphorylating it. GPK-P activates GP (glycogen phosphorylase) by phosphorylating it. GP-P breaks down glycogen into glucose-1-phosphate
==> Inhibition of glycogen synthesis
○ active PKA inactivates GS (glycogen synthase) by phosphorylating it
○ active PKA activates IP (inhibitor of phosphoprotein phosphatase) so now it inhibits PP
Regulation of glycogen metabolism by decreased cAMP
Think: is epinephrine present?
==> Inhibition of glycogen breakdown
○ active PP (phosphoprotein phosphatase) inactivates GPK-P by de-Ping it
○ active PP inactivates GP-P by de-Ping it
==> Stimulation of glycogen synthesis
○ active PP activates GS-P (glycogen synthase) by de-Ping it. GS works to synthesize glycogen
How does CREB relate to memory?
-CREB plays a big role in memory
- When memorizing and repeating info, you start to activate the CREB cycle.
○ Triggers certain neurons in brain to activate certain NTs to signal ===> hyperactivation of CREB which turns on certain genes. And if you turn it on enough, the pathways become self-activating and that’s when memory builds, because of continual exposure to same pathways
CREB signalling pathway (Activation of gene transcription by GPCR)
Example of cAMP and PKA inducing long-term effect (ie, altering gene activity) – slower pathway but longer results.
- genes regulated by PKA have a specific nucleotide sequence known as the cAMP response element (CRE).
- this long-term effect pathway works by activating CREB (CRE-binding protein) transcription factor by phosphorylation of the transcription factor by PKA in nucleus.
- G protein activation via hormone binding GPCR, which activates adenylyl cyclase
- Increase in cAMP levels, activation of PKA in cytoplasm
- The 2 catalytic subunits of PKA translocate into nucleus
- Catalytic PKA phosphorylates CREB
- CREB-P binds as a dimer to cAMP Response Element (CRE) which is on DNA. CREB-P also binds CBP/P300 coactivator (response element). CBP/P300 recruits trancriptional machinery (ie, RNA Polymerase II) —> activates transcription
Phospholipase C and second messengers
- PLC (PLCbeta) is an effector enzyme which is activated by certain Galpha subunits
- Phospholipase C (PLC) cleaves substrate PIP2 (another second messenger) into 2 products: DAG and IP3
- Both these can act as second messengers to amplify certain signalling pathways
IP3/DAG Signalling pathway and Ca2+
- Activation of GPCR by hormone binding, which helps activate alpha subunit and BETA subunit moves along membrane
- Galpha-ATP (active) binds Phospholipase C which allows it to cleave PIP2 into DAG and IP3
- IP3 binds to IP3-gated Ca2+ channel on ER membrane which brings calcium into ER.
- Calcium sends PKC to plasma membrane where it is activated by DAG (2nd pathway)
- So membrane-associated PKC phosphorylates downstream substrates
Smooth Muscle Contraction (NO, cGMP, PKG)
- smooth muscles are found within walls of blood vessels and other organs. If you relax smooth muscle, you open up the blood vessel for more blood flow
Acetylcholine binds GPCR on endothelial cell membrane—>activates PLC, releases secondary messenger IP3 as byproduct of its catalytic rxn –> IP3 binds to IP3-gated Ca2+ channel—> Calcium released from ER –> calcium binds to calmodulin causes activation of enzyme NO synthase, which catalyzes production of Nitric Oxide. - NO binds to NO receptor which catalyses production of anther second messenger cGMP
- cGMP can now bind to the Protein Kinase G — can lead to expulsion of Ca from muscle cells which causes relaxation of muscle cell –> increased blood flow
What’s Calmodulin?
A Ca2+-binding protein which can modulate protein activity
- activator of NO
