Evaluation of SNAP-tag
The application of selectively labeling SNAP-tag-GPCRs to follow ligand-induced receptor internalization was investigated. The use of the SNAP-tag allows researchers to selectively label the specific pool of GPCRs present at the plasma membrane without labeling the internal pools.
To perform this study, the SNAP-tag containing an endoplasmic reticulum import sequence was fused to the N-terminus of various GPCRs (Sig(ER)-SNAP-GPCR). As a consequence, when the SNAP-GPCR fusion protein is inserted into the plasma membrane, the SNAP-tag is exposed to the extracellular environment.
Thus a noncell-permeable SNAP-tag substrate will label only the plasma-membrane-bound SNAP-GPCR proteins.
This selective labeling approach is expected to reduce intracellular background and thus improve the quality of images obtained by microscopic examination of cells.
This holds true for confocal microscopy studies, in which cross sections of cells will be virtually free from signals caused by labeled proteins and also for nonconfocal images where the homogeneous distribution of cell membrane proteins will change to the speckled pattern created by endocytotic vesicles.
The following GPCR model systems were used to investigate the selective labeling of SNAP-GPCRs followed by ligand induced receptor internalization: Neurokinin-1 Receptor and Endothelin-1 Receptor (Gq coupled), b2 Adrenergic Receptor (Gs coupled), and Chemokine Receptor 4 (Gi/Go coupled).
To perform these analysis, cells were transiently transfected by SNAP-tag plasmids using FuGene™ (Roche) according to the manufacturer’s instructions to express the receptor fusion proteins. Fifteen to 24 hours past transfection cells were labeled with the noncell-permeable SNAP-tag substrate BG-488 (fluorescein filter set compatible) at a concentration of 5 µM for 10 min at 37°C in complete medium. Excess substrate was washed away. Subsequently, the appropriate GPCR ligand was added in complete medium and incubated on the cells.
A significant change in the receptor distribution could be observed in all cases within 20 minutes after the start of incubation. Depending on the requirements, the SNAP-tag substrate can be replaced by one, e.g. at 547 nm or at 647 nm excitation wavelength.
Figure 1 shows results for cells transfected with the SNAP-Endothelin-1 Receptor (all images were acquired by nonconfocal fluorescence microscopy). In resting cells, the receptor is homogeneously distributed in the plasma membrane (Figure 1A). Few bright spots can be detected in the cytoplasm indicating low internalization before stimulation.
Post stimulation with endothelin-1 (Figure 1B), the receptor is massively relocalized to endocytic vesicles. Relocalization was clearly detectable at times post stimulation of 10 min at 37°C. Next to the micrographs a drawing depicts the situation before and after stimulation. Only SNAP-tag receptor labeled with noncell-permeable substrate contributes to the relocation observed in that experiment.
SNAP-tag fusions to Neurokinin-1 Receptor (ligand Substance P; expressed in COS-7 cells), Chemokine Receptor 4 (ligand SDF-1a; expressed in COS-7 cells), and b2-Adrenergic Receptor (ligand isoproterenol; expressed in U2-OS cells) also localized correctly to the plasma membrane and were able to internalize into endocytotic vesicles upon ligand stimulation (Figure 2).
As a control, SNAP-GPI, a fusion of SNAP-tag with a protein sequence that leads to the export of the protein to the outer leaflet of the cell membrane followed by transfer of its C-terminus to a glycophosphatidyl inositol anchor, was expressed. Homogeneous plasma membrane labeling was obtained after incubation with BG-488 substrate. Neither in the resting state nor after treatent with any of the ligands used for the GPCRs could any internalization into endosomal vesicles be detected, revealing that ligand-induced endocytosis was specific to binding of the appropriate ligand to its receptor.