Signal Transduction Final Exam 2012



Signal Transduction Final Exam 2012

1A) The researchers attempt to resolve if the two arginine residues (R376/377A) are replaced with alanines in the 1-10 motif affects its ability to bind to caM. Both argines are critical to the amphipathic nature of helix 8 of the 5-HT2C G- protein coupled receptor. The solubilized protein extracts of HEK293 cells was transfected with empty vectors (CTL), or Co-tranfected with wild-type (5-HT2C) and CaM-GFP or mutant receptors (5-HT2CR376/377A). They then treated it with either 1 (M 5-HT (ligand of 5-HT2C receptor) or vehicle for 5 minutes (where Vector represents plasmid and vehicle is solvent). After treatment one of the two, the extracts were co-immunoprecipitated with a polyclonal antibody against GFP. This will recognize and pull out the CaM-GFP fusion protein. Co-immunoprecipitated proteins were analyzed by Western blotting using a monoclonal antibody against GFP and a polyclonal antibody against 5-HT2C.

There are input samples which are no co-immunoprecipitated show 5% of the protein amount used in immunoprecipitation experiments and so no lg signal was observed. The CTL samples show no presence of vehicle, which is an indication that the HEK293 cells won't affect the results. The samples show that the CTL transfected extracts (treated with the solvent), produced no band when treated with anti-GFP-CaM, but produced a band in the Ig molecular weight region when treated with anti-5-HT2C. These results indicate that 5-HT2C and CaM normally bind, and the two arginine residues at R376/377 are essential for them to bind.

b) To ensure the precision of primary antibody, the use of a secondary antibody control would be a negative control for the experiment. The protein extracts would be treated with secondary antibody only during western blotting. The secondary (goat) antibody is against the primary antibody (rabbit). Thus, when treated with any of the extracts (where no primary antibody are used before it), it should produce no signal.

c) An intermolecular FRET would be suitable to confirm this result in living organisms. If coding of plasmids for fusion proteins of CaM-CFP (donor) wild-type (5-HT2C)-YFP (acceptor), and 5-HT2CR376/377A - YFP (acceptor) are made, the fusion will be injected into the same cell, the the cells will be treated with or without 5-HT. If blue colour is observed, this means that they don't bind. In cells with CaM-CFP and 5-HT2CR376/377A - YFP, a blue colour would be seen if no ligand was added (i.e negative control) and ...