Chris

Question 1

Why are CHO cells used? What are the advantages and disadvantages of using recombinant cell systems such as these?

Answer 1

CHO cells can stably express large quantities of recombinant proteins
Adv = stable transfection, authentic post-translational modifications
Disadv = Expensive, difficult to scale up due to complex growth requirement, lengthy expression time

Question 2

What is a point mutation?

Answer 2

Changing a single nucleotide

Question 3

What is sucrose doing, and how?

Answer 3

Sucrose prevents sequestration through inhibition of clathrin-coated vesicle formation. Hypertonic sucrose (0.45 M) causes cells to swell which prevents membrane invagination

Question 4

How does the sequestration data relate to the functional data, and why?

Answer 4

Blocking of sequestration led to no reversal in desensitisation (i.e. no resensitisation)
Wt beta2AR showed resensitisation after agonist-free incubation
Suggesting sequestration of beta2AR is involved in the recycling and resensitisation of desensitised beta2AR

Question 5

What is a splice variant?

Answer 5

Different mRNAs are produced from RNA splicing

The MOR gene has many introns/exons - exons can be ‘spliced’ together in various ways

Question 6

What is monensin doing, and how?

Answer 6

Monensin prevents recycling of internalised receptors back to the plasma membrane
It inhibits acidification of endosomes which prevents dissociation of the ligand from the receptor, meaning the receptor cannot be trafficked back to the plasma membrane so remains intracellular

Question 7

Radioligand binding assays

Answer 7

Quantify how many/proportion of receptors with DAMGO bound

Question 8

Confocal microscopy

Answer 8

Gives information about the localisation of the receptors in the cell