The P{Tub-Arr-TEV.Tango} construct is designed to be used in the Tango-Trace assay, which allows functional synaptic connections that use histamine as the primary neurotransmitter to be traced in vivo. In the histamine Tango-Trace assay, two components are required. The first component, Hsap\HRH2TCS-GAL4.Tub.Tango (carried in P{Tub-HRH2-TCS-GAL4.Tango}), consists of a transcriptional activator (GAL4) that is tethered to the cell membrane by being fused to a G protein-coupled receptor for histamine (Hsap\HRH2), with a specific protease cleavage site (TEVp) interposed between the two coding sequences. The second component, Hsap\ARRB2::TEV\NIaTub.Tango (carried in P{Tub-Arr-TEV.Tango}), is a fusion protein in which the TEVp protease is 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\HRH2TCS-GAL4.Tub.Tango is activated, recruiting Hsap\ARRB2::TEV\NIaTub.Tango and allowing the TEVp protease to access the Tag:CS(TEVp) cleavage site in Hsap\HRH2TCS-GAL4.Tub.Tango. This releases the transcriptional activator (GAL4), allowing it to translocate to the nucleus and activate a reporter construct, providing a stable transcriptional readout of the histamine signaling.
An α-tubulin promoter drives expression of a fusion protein which consists of Hsap\ARRB2 fused to the TEVp protease.
