Advanced Cell Biology II – Homework # 1
Step 1: Determine which GPCRs are associated with the peach scent. (3.5 pts)
Insert the cDNA of all the olfactory GPCRs into E. coli and purify out all the olfactory GPCR proteins.
For each GPCR protein, have one control reaction with just the GPCR, and one experimental reaction where the GPCR is incubated with the peach extract. Do this for all the olfactory GPCRs.
Run all the samples on a gel. If a compound in the peach extract is the ligand for a specific GPCR, it will cause a conformational change in that GPCR. This will cause the band on the gel to shift relative to the control reaction for that GPCR.
This will allow us to identify which GPCRs are associated with the peach scent.
Step 2:…show more content… If GTP cannot be hydrolyzed to GDP, Gɑ-GDP and Gβγ would not re-associate, so the signal will not be turned off.
Cholera and/or Pertussis toxins
Cholera toxin locks Gɑs in the GTP bound active conformation, this would extend the duration of the scent.
Pertussis toxin locks Gɑi in the GDP bound inactive conformation, this would reduce the duration and extent of the scent.
Inhibitors or Activators of Adenylate Cyclase.
Activators of Adenylate Cyclase would cause production of cAMP, which is a second messenger. This would amplify the signal and increase the extent of the scent.
Inhibitors of adenylate cyclase would do the opposite, there would be no and very little cAMP, which would result in little of no amplification of the signal, reducing the extent of the scent.
Inhibitors or Activators of cAMP Phosphodiesterase.
Phosphodiesterase breaks phosphodiester bond and converts cAMP back to AMP, getting rid of second messenger
Activators of cAMP phosphodiesterase would get rid of the second messenger and reduce the extent of the scent.
Inhibitors of cAMP phosphodiesterase would allow the second messenger to exist and amplify the signal, increasing the extent of the scent.
Inhibitors or Activators of a GRK.
Activators of GRK would cause phosphorylation of serine and threonine residues, which can then bind arrestin proteins, preventing reactivation of the signaling pathway.