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Complete Cell Biology Exam Questions & Answers
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Cytoskeleton (Biomedical Science)
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Cell Signalling AllProblem Sheet 1
Olfactory sensory neurons (OSNs) express GPCRs that are activated by the binding of odour molecules,
leading to activation of adenylyl cyclase (AC) by the G olf subunit. Cyclic AMP opens cAMP-gated Ca 2+/Na+
channels in the plasma membrane, leading to depolarisation of the membrane and the production of an
action potential. However, after prolonged exposure to an odour, the neurons stop responding – a
process called adaptation or desensitisation.
Study A
One group of scientists studied isolated mouse OSNs in culture. They discovered that cAMP levels
decrease after prolonged exposure to an odour and that this decrease is correlated with phosphorylation
of AC at Ser1076. Their results are summarised in Table 1.
No odour After brief After prolonged
exposure to odour exposure to odour
Odorant binding to No Yes Yes
GPCR?
Depolarisation of No Yes No
the neuron?
cAMP levels in the Low High Low
neuron?
Phosphorylation of No No Yes
Ser1076 in AC?
Table 1: Summary of results obtained in study A.
Q1-1: Based on these observations, propose a possible molecular mechanism to explain why OSNs no
longer respond to an odour after prolonged exposure.
Upon prolonged exposure to odor, there seems to not be phosphorylation of Ser1076 in Adenlylyl
Cyclase, which could inhibit the coupling with G subunit and prevent it to convert ATP to cAMP, thus
lowering the cAMP levels in the neuron. cAMP is also needed for neuron depolarization so without
cAMP, there is no depolarization. There is some sort of a feedback loop. Something downstream of cAMP
leads to the kinase becoming active that phosphorylates AC and turns down the signalling in the origin.
,Q1-2: What are the likely candidates for the kinase that phosphorylates AC, and how would you test
experimentally which kinase is responsible for AC phosphorylation?
Candidates:
• Protein Kinase A is an obvious candidate because it is regulated by cAMP.
• Calcium-calmodulin dependent kinase (CaMK) is also a possible candidate. The outcome of cAMP
signalling is the opening of ion channels. When the channels open, Na and Ca ions are flowing into
the cell and the membrane depolarizes. Calcium flows into the olfactory sensory neurons, increasing
Ca conc in the cytosol. Search in Summary “Ca2+-induced Ca2+-release”. Calmodulin is activated
which then activates CaMK.
• Regulatory mechanisms in Rhodopsin systems depend on Calcium as well. Cyclic GMP,
phosphodiesterases and the Rhodopsin kinases are also regulated by calcium.
How to test?
To test the mechanism, you need to disrupt the negative feedback loop by inhibiting
multiple kinases and find out which one is involved.
1. Let’s say you think it’s Protein Kinase A
2. Chemically Inhibit Protein Kinase A
3. Could do genetic testing as in knock out the gene coding for PKA. This is more difficult.
Whatever the method, you need to consider that the kinases don’t just have a single substrate. You will
affect multiple pathways and it will be difficult to pin the output to the feedback loop that you’re looking
at.
Q1-3: How would you experimentally determine
(i) Odour binding to the GPCR
Tests for Protein ligand interactions (FRET, Radiolabels) that produce a signal (i.e fluorescence)
when odorant and GPCR come together. For smaller ligands (i.e Adrenaline), there isn’t a way to
fluorescently tag them without disrupting the binding to receptor. Therefore, Radiolabelling >
FRET. Basically,
a. Produce radioactive ligand
b. Expose cells to it
c. Measure the amount of radioactivity associated with the cell membrane at different
concentrations of radioactive ligand
d. You should get a curve that saturates
e. Halfway point = affinity. This is how you know whether or not it’s bound.
(ii) cAMP levels
In the original paper, they used radioactive nucleotides:
1. Fed radioactive adenine to the cells which then became part of the ATP.
2. Lysed the cells
3. Used Ion exchange chromatography to separate the radioactive nucleotides
, 4. There would be peaks in the chromatogram that correspond to ATP, ABP, AMP, cAMP.
5. Because it’s radioactive, it’s sensitive enough to quantify the number of cAMP.
These days, we can use antibodies that quantify cAMP or fluorescence sensors that quantify
cAMP as well. You can put the antibodies/fluorescence sensors into a cell and quantify the
amount of cAMP by the amount of fluorescence.
(iii) AC phosphorylation
In the original paper, they used autoradiography. They used radioactive ATP so that when AC
becomes phosphorylated, they could check how much radioactivity is associated with the band
of AC on an SDS PAGE gel.
These days, we would use Western Blotting. Receptor activation is commonly studied by
Western Blotting using antibodies against phosphotyrosine (anti-pTyr). This is a common
method in which autophosphorylation is detected.
1. Treat the cell expressing the receptor with ligands.
2. Lyse the cell
3. Immunoprecipitate the receptor
4. Run on SDS-PAGE
5. Buy antibodies that would recognize the phosphorylated Ser1076 and blot it on the AC and 5
residues on either side.
6. With increasing amount of ligand, there should be an increase in the phosphotyrosine (or
phosphorylated Ser1076 in this case) signal, which tells us that the receptor has been
activated and becomes auto phosphorylated on tyrosine (or Ser1076). More ligand = more
phosphorylation.
Control: An important control that should always be included is the level of the total receptor in
the immunoprecipitates. To do this, strip the blot and re-probe it with an antibody against the
receptor itself. The control SDS PAGE should show equal loading in each experiment. In this case,
we could conclude that the increase in phosphorylated Ser1076 signal is in fact due to the
phosphorylated Ser1076 itself and not an increased amount of receptor.
Control
, Q1-4: How would you test your proposed mechanism in a living mouse?
The proposed mechanism is:
1. Odorant binds for a long time
2. cAMP levels go up
3. Channels open
4. Somehow there is a negative feedback mechanism linked to AC.
5. Reduces AC activity or its activation via the G protein.
6. cAMP drop
The experimental test would have to prevent the phosphorylation event on Ser1076 on AC. This can be
done by:
1. Producing a knockout of the Protein Kinase A gene. Doing so, the PKA will be inactive and it could
not phosphorylate the AC, so the AC will be constantly active and we will be able to register the
ongoing depolarisation. Again, note that kinases have multiple substrates so this method might
affect other pathways as well.
2. Genetically mutating the Ser1076 to Alanine. If this is successful, the loop would not exist. This
should only affect this specific pathway.
In fact, this method has been done (next qn). There were no significant effects found – mouse
still responded with adaption to odors. Therefore, the conclusion had to be that phosphorylation
at Ser1076 was not the main mechanism whereby adaptation occurs.
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