As a graduate student, you are working on understanding how a new drug regulates

Question

As a graduate student, you are working on understanding how a new drug regulates expression of Gene A. When the cells are treated with the new drug, you notice an increase in phosphorylation of CREB and increased expression of Gene A. Your PhD advisor has asked you to design a series of experiments to determine the type of receptor and signaling pathway involved in this response by cells to the drug.Youve started by treating the cells with a membrane permeable analog of cAMP and noted an increase in phosphorylation of CREB and expression of Gene A. Based on this information, you hypothesize that: (1) the new drug binds to a G a S-coupled receptor to increase intracellular cAMP levels; (2) increased intra cellular cAMP levels are directly related to increased phosphorylation of CREB; and (3) phosphorylation of CREB is required for regulation of expression of Gene A. Explain specifically how you would design experiments using pharmacological manipulations (inhibitors or activators of enzymes along this signaling pathway) or genetic manipulations (knockdown, over-expression,specific mutations, or gene editing of enzymes along this pathway or of CREB itself) to prove eachof these three hypotheses. (Hints: in preparing this question, you should have specific drugs and their targets/mechanisms stated or specific mutations in mind that you might make).

Explanation / Answer

To prove our first hypothesis we can knockdown/mutate the GPCR genes in cells, then we treat these cells and control cells (wild type) with the same amount of drug.In this experiment there should be less intracellular cAMP compared to control cells. similarly we can over-express GPCR genes and treat these cells and control cells with the same amount of drug, but there should be increased level of intracellular cAMP compared to control cells.

To prove our second hypothesis we can stimulate or inhibit adenyl cyclase an enzyme which synthesis cAMP from ATP and compare the phosphorated CREB with the control cells.

To prove the third hypothesis we can do protien kinase assay.

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