Lecture 5

Question 1

Signal transduction

Answer 1

signal transduction converts one form of signal into a different form
external signal (radio waves)  - receptor - transducer - amplifier (radio) - response (sound waves)

Question 2

From signal molecule to target proteins

Answer 2

signal molecule - receptor - intracellular signal molecules - target proteins
receptor-ligand complex activates an amplifier enzyme (extracellular fluid)
receptor located in cell membrane
intracellular signal molecules and target proteins in intracellular fluid
one ligand is amplified into many intracellular proteins

Question 3

Pathway of AC and cAMP

Answer 3

adenylyl cyclase = membrane bound enzyme
cAMP = 2nd messenger
cyclic AMP phosphodiesterase = regulates cAMP levels in cell
5’ - AMP = inactive metabolite

Question 4

Criteria for evoking cAMP

Answer 4

cAMP was invoked to explain responses to a variety of signalling molecules
criteria needed to be set (Sutherland-Rall)
adenylyl (adenylate) cyclase is present
change in concentration of cAMP precedes responses
concentration-response demonstrable
PDE inhibition exaggerates responses

Question 5

cAMP regulation

Answer 5

phosphodiesterases (PDEs) regulate levels of cAMP/cGMP
multiple isoforms exist
11 different families
any single cell can express different PDEs
selectivity of PDEs targeted by drugs
modulate the amount/temporal changes in levels of these nucleotides
inhibition can have profound effects (eg. sildenafil)

Question 6

Unanswered questions

Answer 6

what intervenes between the recognition of a messenger molecule by the receptor and the activation of adenylyl cyclase to produce cAMP?
how does cAMP itself produce effects?

Question 7

Amplification cascade

Answer 7

initial stimulus activates the first step in the cascade
catalytic subunit activates the first enzyme in cascade
conversion of substrate to product is the final step of the cascade
reception (ligand binds to G protein-linked receptor) = 1 molecule
transduction
response
eg. glycogen - glucose (10^8 molecules)

Question 8

Transduction

Answer 8

inactive G-protein - active G-protein = 10^2 molecules
inactive adenylyl cyclase - active adenylyl cyclase = 10^2 molecules
ATP - cAMP = 10^4 molecules
inactive protein kinase A - active protein kinase A = 10^4 molecules
inactive protein - phosphorylated (active) protein = 10^6 molecules
inactive protein - phosphorylated protein = 10^6 molecules

Question 9

Reversible phosphorylation

Answer 9

target protein inactive - target protein active
phosphorylation (via kinases)
dephosphorylation (via phosphatases)

Question 10

Pathway of phosphorylation

Answer 10

signal molecule binds to G-protein linked receptor, which activates the G-protein
G-protein turns on adenylyl cyclase, an amplifier enzyme
adenylyl cyclase converts ATP to cyclic AMP
cAMP activates protein kinase A protein kinase A phosphorylates other proteins, leading ultimately to a cellular response

Question 11

Receptor to adenylyl cyclase

Answer 11

G-protein coupled receptor and adenylyl cyclase enzyme are two separate entities embedded in the cell membrane
G-protein is comprised of 3 subunits (alpha, beta, gamma), making it a trimeric protein, and is bound to GD

Question 12

G-protein sequence

Answer 12

signalling molecule binds to active site of receptor
conformation change in the intracytoplasmic loop allows for binding of G-protein (trimeric structure)
alpha subunit exchanges its GDP for GTP
the trimeric protein dissociates
alpha subunit with attached GTP activates adenylyl cyclase
after activating AC, unstable alpha subunit dephosphorylates GTP to make it GDP, beta and gamma units reattach
activated AC converts ATP to cAMP
cAMP binds to regulatory subunit on protein kinase A to allow the catalytic subunit to act

Question 13

Protein-kinase activation

Answer 13

catalytic subunit activates the first enzyme in cascade
activated enzyme phosphorylates another inactive enzyme to activate it
this is done by transferring a phosphoryl group from ATP
the activity of the first activated enzyme is terminated by dephosphorylation
the second activated enzyme phosphorylates another inactive enzyme
amplification occurs as sequence continues
phosphorylation-dephosphorylation sequence is crucial

Question 14

Histamine receptors

Answer 14

H1  = Gq/11 (increase IP3/DAG)
H2 = Gs (increase cAMP)
H3 = Gi (decrease cAMP)
H4 = Gi (decrease cAMP)

Question 15

Other second messengers

Answer 15

Ca 2+
diacylglycerol
phosphoinositide
inositol triphosphate
same principles apply in identifying these mediators in cellular function
considerable cross-talk between 2nd messengers

Question 16

Other receptors

Answer 16

extracellular signal molecules
ligand gated channel = ligand binding opens or closes the channel
ligand binding to a receptor-enzyme activates an intracellular enzyme
ligand binding to a GPCR opens an ion channel or alters enzyme activity
ligand binding to integrin receptors alters the cytoskeleton
anchor protein