Protein fusions to the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), the so-called SNAP tag, have been used to construct a variety of assays. Assays for endocytosis have typically involved high-content imaging approaches using green fluorescent protein (GFP)-tagged membrane receptors. The work described here uses an O6-benzylguanine (BG) linked to a fluorophore via a disulfide bond.
Labeling of a cell surface receptor fused to a SNAP-tag allows monitoring endocytosis by removing the surface bound fluorophore with a cell-impermeable reducing agent such as tris(2-carboxyethyl)phosphine (TCEP). Following TCEP treatment of cells, only the internalized fluorophore is monitored (see Figures A–D). The endosomal environment is thought to be generally nonreducing, and so the internalized BG-S-S-488 probe is not cleaved in the endosome.
To test this, the authors used a cell permeable form of TCEP (a trimethyl ester analog, tmTCEP). No free dye could be extracted from cells containing endosomes with SNAP-β2-adrenergic (ADR) which were labeled with BG-S-S-488 when TCEP was used; however, free dye was extracted from these cells when tmTCEP was used. Therefore, the normal environment of endosomes is apparently nonreducing. Endocytosis and recycling can be quantified with this method because the same cells can be used to measure the total fluorescence from labeled receptors on the surface (before treatment with TCEP) as well as the internalized fluorescence after treatment with TCEP.
A two-color assay is also shown in this article in which an RFP-tagged β2-arrestin, and either SNAP-β2ADR or SNAP-NK1R are imaged. Agonist treatment shows translocation of the RFP-β2-arrestin2 to the cell membrane, and following TCEP treatment the co-localization of the receptors into endosomes could be measured. With this method different populations of receptor/β2-arrestin complexes can be studied.
The assay can be applied to any cell surface protein and could be expanded to study other internalization processes such as phagocytosis as well.
Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.