Question

2. The cell line of S49 lymphomas is commonly used to study adenylate cyclase. There Are cells with mutations in some of the system components. The Wild variant of S49 expresses receptors for the hormone epinephrine. The following graph illustrates measurements made using membranes from a S49 mutant line. The data in the graph on the left represent wild cells incubated only with GTP (with) with epinephrine and GTP (EPI) or treated 1 hour earlier with the cholera toxin, GTP and NAD + (CT). At time 0 in the graph, radioactive ATP was added at the beginning of the experiment. The Central graph is a repetition of the experiment using mutant cells. The graph at the end shows the link of epinephrine to the wild (square) and mutant (triangles) cells. Wild Type Variant Binding 1000 1000 CT 750F 2500 E 500 epi 250 con con epi 0 0 5 10 15 20 0 5 10 15 20 0 100 200 00 Epinephrine] (nM) Minutes Minutes A Based on the data choose which (s) of the following mutations may be the (n) observed in the mutant cells. Explains each why it is or is not a possible mutation. (2 points each) . Mutation in one of the cytoplasmic domains of the B-adreniergic receptor thatis linked to Gs. . B-adrenergic Receptor with reduced affinity to epinephrine Mutated Gs Protein that cannot be associated with the receptor . Mutated Gs Protein that cannot be associated with adenylate cyclase. . Mutated Gs Protein that cannot bind GTP

Answer 1

To understand the answer, you should understand the strategy first. You know epinephrine is a hormone that binds adrenergic receptor. These are G protein-coupled receptor (GPCR). Do you know what G-protein coupled receptor does? After binding of a specific hormone, GPCR gets associated with G-protein and activates it by exchanging its GDP for a GTP. GTP bound G-protein then dissociate from membrane receptor. Adenylate cyclase gets activated by G-protein. After activation, it produces CAMP from ATP.

Now, look at the figure. If you see the graph no 3, you can find that both wild type and mutant cell has almost same binding capacity with Epinephrine. So, binding is normal for both of them. Look at graph 1. This is so obvious for a wild cell, that addition of epinephrine and GTP (Epi) will yield more cAMP than control condition (con), where only GTP has been provided, but no epinephrine is added. And when you are providing cholera toxin (CT), cAMP production is high. But why? What does CT do? Look at graph 2. As the cell is mutated, it is so obvious that cAMP production is less for both the cases (con and Epi). But here also production of cAMP is very high in case of CT. Actually CT does ADP ribosylation to the G-protein. What does it mean. ADP ribosylation is a process where ADP is added in G-protein by using NAD+ as a cofactor. By doing this, it keeps that G-protein in an activated state (GTP bound). So, cAMP production is higher.

Now, coming to your choice,

Mutation in one of the cytoplasmic domains of the B-adrenergic receptor that is linked to Gs.

This is not correct. As, CT can not modify that part.

B-adrenergic receptor with reduced affinity to epinephrine.

This is not correct. Because from graph 3, you can see that receptor has a strong affinity to epinephrine.

Mutated Gs protein that cannot be associated with the receptor.

This is not correct. Because CT can not modify the mutated protein in this way that an non-associated protein can associate receptor.

Mutated Gs protein that cannot be associated with adenylate cyclase.

This is not correct. Because G-protein could not bind adenylate cyclase even after the addition of ATP, so no cAMP would produced then.

Mutated Gs protein that cannot bind GTP.

This is correct. CT modifies the G-protein in this way that it can bind ATP now forever. Because, it can not hydrolyzes ATP into ADP. So, ATP bound G-protein can produce cAMP in high amount.