Fluorogen-activating proteins – FAPs – are polypeptides that bind organic fluorogen molecules at nanomolar concentrations to yield fluorescent complexes. Neither the FAP nor the fluorogen is fluorescent by itself.

Agonist stimulation of G-protein coupled receptors (GPCRs) typically triggers receptor internalization. FAP-based assays are particularly useful for detecting and quantifying this response. For these assays, receptors are expressed with FAP tags at their extracellular N-termini.

When cells expressing FAP-tagged receptors are exposed to membrane impermeant fluorogens, receptor molecules at the cell surface acquire fluorescence but receptor molecules inside the cell do not. If fluorescence subsequently appears inside the cell, it must come from receptor molecules that were internalized after fluorogen addition. 

SpectraGenetics offers two powerful FRET-based assays that detect and quantify receptor internalization.

pH-FRET Assay

The pH-FRET assay employs a fluorogen linked to a pH-sensitive Cy3 fluorophore. When bound to the FAP, this molecule yields red and FRET fluorescence.

The FRET signal – but not the red signal – is enhanced at acidic pH, and so receptor molecules that have been internalized give higher FRET signal than receptor molecules at the surface. This enables one to quantify the fraction of receptors that have been internalized by measuring FRET-to-red ratios in living cells.

In brief, the assay is performed as follows:

Step 1. Add fluorogen and test compound to cells expressing FAP-tagged receptor

Step 2. Incubate at 37 degrees

Step 3. Measure signal in red and FRET channels during incubation to detect and quantify receptor internalization

The pH-FRET assay is a real-time live-cell assay that gives kinetic output. An example is shown below for isoproterenol stimulation of cells that express a FAP-tagged human adrenergic receptor (ADRB2). Direct fluorescence is red, and FRET signal is green.

SS-FRET Assay

The SS-FRET assay employs an alpha2 fluorogen linked to a Cy3 fluorophore by a linker that contains a disulfide bond. When bound to the FAP, this molecule yields red and FRET fluorescence.

Treatment of cells with a membrane-impermeant reducing agent eliminates FRET signal from receptors at the cell surface but does not affect FRET signal from internalized receptors. This enables one to quantify the fraction of receptors that have been internalized by measuring FRET-to-red ratios.

In brief, the assay is performed as follows:

Step 1. Add  fluorogen and test compound to cells expressing FAP-tagged receptor

Step 2. Incubate for 10-40 minutes

Step 3. Add reducing agent

Step 4. Add fixative

Step 5. Measure signal in red and FRET channels to detect and quantify receptor internalization

Because cells are fixed before measurement, the SS-FRET assay allows one to separate the wet-lab steps from the analytical steps of the assay. Example results are shown below for HEK 293 cells expressing the human gastric inhibitory polypeptide receptor (GIPR). Compared to controls, cells treated with agonist (5 micromolar GIP for 40 minutes) show a nearly twofold increase in the FRET-to-red ratio (0.75 versus 0.38) indicating that approximately 50% of the surface receptors were internalized.