Figure 1.

Construction and expression of SNAP- and CLIP-tagged forms of the human OX₁ and CB₁ receptors. (A) Upper panel: cartoon representation of the signal/leader sequence, an epitope tag and the SNAP or CLIP sequence within the N-terminal extracellular domain of a 7 transmembrane domain GPCR. A schematic cartoon of the basis of selective covalent modification of proteins that incorporate SNAP or CLIP tags is also shown. The fluorophore or other label becomes linked permanently via the O⁶-alkylguanine-DNA-alkyltransferase activity of the SNAP (SNAP-tag substrates consist of dye conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker)/CLIP (CLIP-tag substrates consist of dye conjugated to a cytosine leaving group via a benzyl linker) proteins. Lower panel: Cartoon representation of the use of SNAP- or CLIP-Lumi4Tb to label cell surface SNAP/CLIP-tagged GPCRs. Excitation with light of 337 nm results in long-lived fluorescence output at 620 nm that can be harnessed to a variety of homogeneous time-resolved fluorescence assays. (B) Lysates prepared from uninduced (− dox) or construct-induced (+ dox) cells that harbour VSV-G-SNAP-OX₁, VSV-G-SNAP-CB₁ or HA-CLIP-CB₁ were resolved by SDS-PAGE and subsequently immunoblotted with anti-VSV-G, anti-HA or anti-SNAP/CLIP (which recognizes both sequences).