An α-tubulin promoter drives expression of a fusion protein which consists of Hsap\HRH2 fused at its C-terminus to Scer\GAL4, with the two coding sequences interposed by a TEV protease cleavage site (Tag:CS(TEVp)). The presence of Hsap\HRH2 (a G protein-coupled receptor) tethers the fusion protein to the cell membrane. The GAL4 driver can be released from the membrane by cleavage of the Tag:CS(TEVp) site with the TEVp protease.
Designed to be used in the Tango-Trace assay, which allows functional synaptic connections (which use histamine as the primary neurotransmitter) to be traced in vivo.
In the histamine Tango-Trace assay, two components are required. The first is Hsap\HRH2Tub.Tango.T:Scer\GAL4 (carried in P{Tub-HRH2-TCS-GAL4.Tango}), which consists of a transcriptional activator (Scer\GAL4) which is tethered to the cell membrane by being fused to a G protein-coupled receptor (Hsap\HRH2), with a specific protease cleavage site (T:Zzzz\TEV.CS) interposed between the two coding sequences. The second component is Hsap\ARRB2::Zzzz\NIaTub.Tango (carried in P{Tub-Arr-TEV.Tango}), which is a fusion protein consisting of the protease (Zzzz\NIa) that can cleave the site present in the first component fused to a cytosolic signaling protein (Hsap\ARRB2) that interacts only with the activated form of the G protein-coupled receptor present in the first component. In the presence of the histamine ligand, the G protein-coupled receptor in Hsap\HRH2Tub.Tango.T:Scer\GAL4 is activated, recruiting Hsap\ARRB2::Zzzz\NIaTub.Tango and allowing the Zzzz\NIa protease to access the T:Zzzz\TEV.CS cleavage site in Hsap\HRH2Tub.Tango.T:Scer\GAL4. This releases the transcriptional activator (Scer\GAL4), allowing it to translocate to the nucleus and activate a reporter construct, providing a stable transcriptional readout of the histamine signaling.