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.

1. 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?

Answer 1

In order to ensure the coupling of the ligand with an appropriate receptor, say Gs or Gi, a very widely used technique is in-situ hybridization. This technology uses the binding of a chromophore (or fluorophore) attached probe (generally a complementary sequence to the target DNA or protein) and allows them to react in vitro/in vivo for a sufficient period of time. After a sufficient incubation, the chromo-/fluore-phore is allowed to react with an appropriate substrate to give either an insoluble colored product or emits fluorescence. This signal can be easily recorded on a recording system, say a fluorescent microscope or a spectrophotomter.

Thus, one can design these fluorophores complementary to the target receptor, say Gs or Gi, allow them to react and examine the results. This will give a very specific result to which receptor the ligand binds to.