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 an agonist molecule called “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 ,G77R, which is expressed in the very brain regions you see effects with “excitor”. You are convinced this cDNA may represent the cDNA for the receptor for “excitor”, the discovery of which could lead to a promotion and pay increase for you.

Describe how you will demonstrate that this cDNA G77R encodes a specific receptor for “excitor” by performing a specific receptor binding assay on whole cells. Include which cells you will use, your controls and brief descriptions of the methods used.

Just looking to understand what it means by specific receptor binding assay... would a radio labeled ligand receptor assay be an option? Would using whole cells change what assay could be used? Or is a pull down assay an option since the potential receptor is a GPCR? If so, would a pull down assay be considered an example of a specific receptor binding assay? The G77R is just a name given to the potential receptor.

Answer 1

The radiolabeled ligand binding receptor binding assay would be the better option for the present scenario. Radioactive ligands are commonly used to measure ligand binding to receptors.​ In this assay either whole cell or cell membrane containing receptor can be used. Radioligands with high specific activity are well suited for the assy. For beeing hidrophobic ligand the non specific binding would be avoided using BSA while washing steps. The highly pure ligand and highly selctive ligand would give specfic binding. Radioactive ligand binding assays can be performed is several different formats mostly it would be performed in filtration format. The assay can also be performed in SPA formats, or in FlashPlates. SPA and FlashPlate formats are homogeneous formats, which means no wash steps are required. In the filtration assay, you will wash away unbound ligand using a vacuum manifold or cell harvester.

​The radioligand will be allowed to bind to the receptor either on the whole cell or on the membrane. Later filtered through filtermat or Unifilter plate usig a cell harvester , filters are washed to remove unbound radio ligand. Filtermat need to be dried and scintillationncocktail is added and data will be read in appropriate detector.