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1. Receptors are critical to cell function. On October 10, 2012, Robert J. Lefkowitz, M.D., (an alum of Bronx High School!) of Duke University Medical Center and Brian K. Kobilka, M.D., of Stanford were awarded the Nobel Prize for their work on GPCRs. At the current time we know of 1000 genes that code for GPCRs.
a. What particular receptor type did these two scientists study for which they are now famous?
G-protein coupled receptors that transport outside stimulus to the inside of a cell
b. What is the role of the "G protein" in GPCRs?
It transports outside stimulus from the outside to the interiors of a cell
c. One important receptor type is the GPCR that is activated by acetylcholine referred to as the muscarinic acetylcholine receptor. How is it distinguished from the nicotinic acetylcholine receptor?
d. What would happen if GMP-PNP were added to some isolated cardiomyocytes that you are using for studying the muscarinic receptor? (Assume that you are able to get this compound into the interior of the cell).
e. The term "orphan receptors" is often used when referencing the GPCR. What does the term orphan mean in this case?
f. Sketch a hydropathy plot of a typical GPCR.
g. One of the more interesting of the GPCR systems is that of light activated rhodopsin. In this system what is the GPCR and what is the target and function of the G protein?
h. How can G proteins interact with adenylyl cyclase to both increase and decrease cAMP concentration in cells?

2. Second messenger systems come in a variety of different forms but they all share in common that their end game is to transmit the fact that a ligand has bound to the receptor that is associated with the second messenger system.
a. What is the function of protein kinase A and how do the regulatory units control its function?
b. The protein kinase A catalytic unit referenced above can have both a cytosolic as well as a nuclear effect. describe how this is the case.
c. Membrane soluble versions of EGTA and EDTA, both calcium chelators, are commercially available. When you use these agents you find that a particular second messenger event is blocked. What might be the reason for this observation?
d. The nitric oxide system is different than most other second messenger systems because it can affect several cells outside of the cell that is originally activated. What is the basis for this phenomenon?

3. EGF (Epidermal Growth Factor) is a ligand that once bound to its target receptor tyrosine kinase often triggers cells to divide.
a. Why is "dimerization" important in this process?
b. Often patients with breast cancer are considered HER2 positive or negative. What is the significance of this designation?
c. Kadcyla was FDA approved in 2013 for treating late stage, metastatic, breast cancer. It is considered a member of a new and emerging group of conjugated antibodies. How does it work?
d. Why does the word "autophosphorylation" relate to the EGF receptor?
e. What is "ras" and why is it important when considering cancer cells?

 

4. The cell has a variety of methods that determine if a protein becomes an integral membrane protein or if it is secreted. The following problems relate to this group of processes.
a. What is the difference between constitutive and regulated secretion? If you were tracking the secretion of insulin which path would it most likely follow?
b. What is the significance between t-SNARE and v-SNARE and why are they important in protein trafficking?
c. You suspect that the protein that you are researching is glycosylated with fucose in the medial Golgi. How could you determine if this is the case?
d. What is the role of pH in KDEL peptides that are mis-sorted to the cis-Golgi?
e. You identify a soluble cytoplasmic protein that has a unique targeting sequence that you think directs it to the mitochondrion. Once there, you presume that this targeting sequence is then cleaved off. How might you investigate this experimental situation and determine if its translocation into the mitochondria is a receptor mediated event due to the presence of a receptor on the mitochondria outer membrane?

5. The Golgi apparatus and the Trans Golgi Network (TGN) is an interesting continuum of two related organelles that sort and modify proteins once they have been synthesized.
a. How was temperature important to the ultimate discovery and elucidation of the TGN?
The temperature, which was twenty degrees Celsius, was so important in the original discovery of the TGN because it was at this temperature that no budding occurred at the TGN but yet the proteins were transported from the ER to the Golgi. This is what made the TGN grow so large in size and made it visible.

b. Mannose-6-phosphate receptors (M6P-R) are found in the TGN as well as on the outer plasma membrane. Both bind newly synthesized lysosomal proteins and help them sort to the lysosome. What is the importance of the M6P-R on the outer cell membrane?
The M6P acts as an address tag on the lysosomal proteins throught the N-acetylglucosamine transferase. The
c. How could you show that the lysosomal hydrolytic enzymes work best at a pH of 5.0 in contrast to 7.0?

6. The Unfolded Protein Response (UPR) is a necessary component of protein regulation and degradation.
a. What is the purpose of the UPR and how does it determine whether a protein will be processed in the RER or discarded?
b. Detail the steps a protein would take from initial stress to degradation as part of the UPR.
c. Why might a scientist or doctor want to inhibit the UPR? Are there any inhibitors that may aid in blocking the UPR pathway?

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