Question

You are asked to divert resources in your research division to fund a project to identify the receptor for “excitor”, a plant derived compound which is very hydrophobic, but potent at stimulating animal neurons to grow. Your research group has been studying the effects of “excitor” in a rat model for anxiety. The agonist is potent at inhibiting anxiety, so could potentially lead to a useful therapeutic for anxiety disorders. Your company has a library of putative GPCR cDNAs for which they also have RNA expression data in numerous rat tissues. After painstaking analysis on your computer, you have found a candidate GPCR cDNA which is expressed in the very brain regions you see effects with “excitor”—you are convinced this cDNA may represent the cDNA for “excitor”, the discovery of which could lead to a promotion and pay increase for you.

7. You believe that excitor can affect the levels of cAMP in the cell. Describe an assay to measure accumulation of cAMP in a cell. Why might you include forskolin? Why might you include a phosphodiesterase inhibitor?

8. You may now assume that you have established that a labeled version of “excitor” binds specifically to the expressed receptor. To confirm that this excitor receptor is indeed a G protein-coupled receptor that requires G proteins to affect downstream functions, how would you experimentally demonstrate receptor coupling through each of the G proteins, Gs and/or Gi?

9. Briefly describe 5 approaches you might use to specifically inhibit the function of a protein in a cellular signaling pathway.

Answer 1

7.A cAMP accumulation assay developed by Creative BioMart is a platform optimized for the detection of a specific range of cAMP concentrations, to enable assay sensitivity fine tuning. It gives high quality, cell-based assay screening in 96-, 384- or 1536-well formats Detection of cAMP is based on the competition between cAMP produced by cells and a biotinylated cAMP probe that is recognized by the streptavidin-Donor and anti-cAMP conjugated Acceptor beads. The beads are brought into proximity and a signal is detected. Increased intracellular concentrations of cAMP following Gs coupled GPCR activation by an agonist results in the displacement of the biotinylated cAMP probe and leads to a proportional signal decrease. The effect of antagonists and reverse agonists can similarly be detected. Gi coupled receptor activation can be detected after prestimulating cells with forskolin.

Concentration-response assays were performed by Maria Rosario Tubio for the studyof expression of GPCR leads to attenuation of signaling by other GPCRs gives a cAMP assay method as follows: Incubate the cells for 3 min in culture medium supplemented with 1 mm IBMX at 37 °C, followed by a 7-min exposure to different concentrations of ligands. The reaction was stopped by the addition of ethanol. The ethanolic phase was then dried and the residue resuspended in 50 mm Tris-HCl, pH 7.4, 0.1% bovine serum albumin. cAMP content was determined by competition of [3H]cAMP for protein kinase A.

Forskolin

The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific adenylate cyclase activator, to increase the basal cAMP level in cells to create an assay window for ligand detection.

The cyclic nucleotide phosphodiesterases comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains. PDEs are therefore important regulators of signal transduction mediated by these second messenger molecules. Specific phosphodiesterase inhibitors block the inhibition of cAMP accumulation.

9. A signal pathway is important for cellular activities like growth, death and division of cells. Signal transduction inhibitors block signals passed between molecules. In chemotherapy, various drugs are used to arrest the cell division. Imatinib is the first protein kinase inhibitor to be approved for cancer treatment.