Receptor Re-Sensitization Assay Using Fluorogen Activation Peptides (FAPs)

A direct, homogenous re-sensitization assay can be run on the same cell line that was used for an agonist or antagonist assay.  Again a very simple  protocol can be used:

  1. Add agonist, red fluorogen and allow time for respone
  2. Add green fluorogen (optional – wash out agonist)
  3. Add test compound (resensitizer-changes rate of receptor re-sensitization) and allow time for response
  4. Read

This assay allows for a number of interesting measurements including:

  1. Rate of receptor recycling (re-sensitization) as a function of agonist
  2. Rate of receptor recycling as a function of antagonist (with or without agonist present)
  3. Rate of receptor recycling as a function of re-sensitizer (with or without agonist present)

An example of the re-sensitization assay readout is shown below. The increase in green signal (decrease in red) represents the return of internalized protein to the cell surface (receptor re-sensitization)

Resentization

 

At about 60 minutes the agonist was washed out and the receptor rapidly returned to the cell surface. The rate of return can easily be measure for different agonists, antagonists (in the presence of agonist and without) and re-sensitizers (in the presence of agonist or without).  SpectraGenetics has tagged more than 150 GPCRs and has validated more than 30 cell lines utilizing the fluorogen activating technology.

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