Question

About 30 different bitter taste receptors in mammals have been identified (i.e., their DNA sequences are known). These receptors can sense a wide variety of poisonous chemicals. Please describe an experiment in cells to find out which receptor is responsible for the sensing of a particular poisonous chemical (let’s call it Y).

Answer 1

In order to find out the receptor responsible for sensing of a particular poisonous chemical, say Y, we can design the following experiment:

First let us understand the basis of our experiment. There are about 30 different receptors whose DNA sequence we know. Also each receptor specifically binds to a particular chemical. So we have to use this ligand- receptor interaction to isolate the receptor first and then analyse it. Now how do we do that.

Since we know the DNA sequences of our receptors we first need to clone all the receptor DNA sequences and add a GFP ( Green Floroscence Protein) tag to it during the cloning process. We first have to amplify the receptor sequences by Polymerase chain reaction with primers, having GFP sequence at its 5' end (so that the receptor becomes GFP tagged when expressed). Then we have to transfect cells that normally don't synthesize these receptors with the amplified PCR products, cDNA for each receptor transfected seperatly and the transfected cells are properly marked and allowed to grow.The GFP tagged receptor proteins will be expressed in the cells that have taken up the cDNA. These cells can then be allowed to bind to radiolabelled Y molecules. The cells that actually expresses receptor for Y molecules can be detected based on the radioactivity within the cell (as only cells expressing the receptor for Y molecules will take them up and interaction within cell will occur) and the ligand receptor complex may be pulled down using an anti-GFP antibody and the amino acid sequence of the receptor determined. Hence we can determine which receptor binds to the Y molecules.