Physiology 3140A Lecture Notes - Lecture 20: Adenylyl Cyclase, Gs Alpha Subunit, Protein Kinase A
2 May 2018
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Cell Physiology Lecture 20
Cyclic AMP and PKA
November 3 2017
- PM is made up of a lot of membrane lipids (phosphatdyllipids) that are degraded through a series of
different enzymatic reactions to create a wide range of substances
o PKC activation + regulation
o Ca++ is a signalling molecule in the cell; regulated through the inositol phospholipid
pathway
Cyclic AMP (cAMP)
- Synthesized from ATP by plasma membrane-bound adenylate cyclase
- Degraded to 5'-AMP by cyclic AMP-specific phosphodiesterase
o cAMP is degraded to 5’ AMP
o This specific phosphodiesterase is able to open up the ring
o LEAVES THE PHOSPHATE GROUP ON THE MOLECULE
o When the ring is open, the molecules does not have the same energy associated with it
- Manufactured from ATP (adenosine tri phosphate)
o Adenosine moiety
o Tri phosphate moiety; 3 high energy phosphate molecules
- Adenylate cyclase (membrane bound enzyme) can chop off the phosphates
o End up with a monophosphate (AMP)
o It becomes cyclized – ring like structure on the molecule (it stores a lot of energy in that
ring)
▪ Signalling molecule in the cell
- ATP is NOT a cell signalling molecule within the cell
o ATP can serve as a ligand for many receptors (purinergic receptors)
▪ Purinergic receptors is a large class of receptors; ATP + ATP analogs can serve as
ligands but not considered signalling
o Used for other purposes: energy generation, substrate for adenylate
cyclase to make cAMP
- 5’ AMP does not have any biological activities in terms of the signalling
molecule in the cell
o Once the ring structure is broken, you get a molecule w/o biological
activities
- cAMP is a ligand; ability to signal into the cell
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cAMP as a signaling molecule
- To function as an intracellular messenger, cAMP concentration (normally < 10-7 M) must be able to
change up or down rapidly in response to extracellular signals
- Has to be present in a normal resting cell the cAMP concentration is VERY low – 0 to 10-7M
- For something to serve as a signalling molecule, it has to be present in a resting cell at 0 to low
concentration
o In normal resting cells, the cAMP concentration is VERY LOW
- To become a signalling molecule, it has to be able to change its concentration VERY quickly
- Step wise function:
o In a normal resting cell, have basal cAMP level (extremely low) that is below a level that can
mediate any biological effect in the cell
o Cell receives information (get a ligand binding to a receptor get activation of adenylate
cyclase), get synthesis of cAMP
▪ [cAMP] goes up VERY rapidly
o It remains high for a certain period of time until phosphodiesterase degrades
o [cAMP] goes back to the basal level
- The step wise function is what signals to the cell to mediate a biological effect
- Example: Upon hormonal stimulation, cAMP levels can change 5-fold within a few seconds
- The change of cAMP does not have to be huge
o Can be 5-10 fold
o For example, a change from 10-7 to 10-6 can mediate a biological effect
o Then it falls back as the phosphodiesterase cuts it down – goes back to resting levels
- What is signalling when you have a low level, goes up 5-10 fold, and then it mediates an effect?
o What happens with that increasing concentration? SIGNAL AMPLIFICATION
o What is the immediate thing that happens: There are more molecules to activate PKC
- Initial step: since there is an increase in concentration of the second messenger, the probability that
there is going to be a biological effect is going to increase
o Signal amplification, changes in gene transcription, etc.
- Relates to the ability of the second messenger to bind to its targets (ex. PKA)
o Relates to the affinity for binding – now the probability has increased, because getting into
the range where binding affinity is appropriate for that phenomenon to occur
Primary physiological function of cAMP
- Serve as an intracellular second messenger to bind to and activate protein kinase A (PK-A)
- Act as a ligand for a specific class of odorant cation channels in olfactory neurons
o cAMP can also be a ligand to bind a specific group of cation channels that detect certain
odours
o Ligand operated ion channel on the inner surface of the plasma membrane, & the ligand is
cAMP
o cAMP is generated binds to the ion channel causing a change in the gating properties of
the ion channels lead to function in olfactory neurons
- cAMP can be a ligand to bind PKA to mediate an effect
- Ligands can be both extracellular and intracellular; binding to a particular receptive site
- In sensory organs; cyclase AMP can serve as a ligand!
o Ligand to bind to PKA to mediate an effect
- TWO FUNCTIONS OF cAMP: 1) 2nd messenger to activate pKA 2) ligand for odorant cation channels
find more resources at oneclass.com
find more resources at oneclass.com
Adenylate cyclase
- Consists of two alternating hydrophobic and hydrophilic domains
- Hydrophobic domains each contain 6 membrane-spanning domains
o It is like a duplicated molecule
o 6 transmembrane domains – catalytic domain – 6 transmembrane domains – catalytic
domain
o There are two hydrophobic domains!
- Hydrophilic regions having two catalytic domains
- Multiple isoforms of adenylate cyclase - stimulated by Gs and inhibited by Gi
o GPCR couples to a G protein:
▪ Gs- stimulation of adenylate cyclase
▪ Gi – inhibition of adenylate cyclase
o Alpha subunit is going to interact with adenylate cyclase leading to modulation of its activity
- Adenylate cyclase is a membrane bound enzyme (found at the plasma membrane mostly) that
generates cAMP
o Note: this is a family of enzymes, therefore there are variants that can be formed
- Biological amplification: Ligand-receptor complex with G protein. Engaging an effector, which is a
double molecule (two catalytic domains) that can amplify the signal
o Gone from a single situation to amplification
- TWO HYDROPHOBIC DOMAINS + TWO CATALYTIC DOMAINS
o Alternating!
- Single enzyme molecule!
Cyclic AMP-dependent protein kinase (PKA)
- Inactive PK-A consists of a heterotetrameric complex with 2 catalytic subunits and 2 regulatory
subunits
- Increase in cAMP in response to activation of adenylate cyclase
- 2 cAMP molecules bind to each regulatory subunit leading to conformational rearrangement and
dissociation from the complex
- Ligand binds to the receptor couples G- s turns on the effector (Adenylate cyclase) cAMP
generated
- Note: Adenylate cyclase makes cAMP
- cAMP was at a low concentration in the cell (≤-7M). Get a jump within a matter of seconds
o During the step function, cAMP is at a concentration where the probability that it to going to
bind to sites that have affinity for it, can take place
▪ Think about KM (the binding affinity)
▪ KM for the cAMP to bind to PKA is going to in the range of 5-7M
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Synthesized from atp by plasma membrane-bound adenylate cyclase. Manufactured from atp (adenosine tri phosphate: adenosine moiety, tri phosphate moiety; 3 high energy phosphate molecules. Adenylate cyclase (membrane bound enzyme) can chop off the phosphates: end up with a monophosphate (amp) It becomes cyclized ring like structure on the molecule (it stores a lot of energy in that. To function as an intracellular messenger, camp concentration (normally < 10-7 m) must be able to change up or down rapidly in response to extracellular signals. Has to be present in a normal resting cell the camp concentration is very low 0 to 10-7m. For something to serve as a signalling molecule, it has to be present in a resting cell at 0 to low concentration. In normal resting cells, the camp concentration is very low. To become a signalling molecule, it has to be able to change its concentration very quickly.
