Figure 8

Nrg1 induced interactions of ErbB4 and adapter proteins. (a) Schematic drawing showing the principle of the assay. Upon Nrg stimulation, ErbB4 receptors are activated and Tyr-phosphorylated resulting in binding domains for several adapter proteins such as the PI3Kp85 subunits. In the split-TEV assays, ErbB4 is fused to N-TEV, a TEV cleavage site and the transcription factor GV (ErbB4-N-TEV-GV). The adapter proteins are fused to C-TEV (such as PI3Kp85-C-TEV). PI3Kp85 binding to ErbB4 results in the formation of a functional TEV protease made up of the N-TEV and C-TEV fragments. Proteolytic activity releases GV, which can translocate to the nucleus to induce transcription of a luciferase reporter gene. (b) Schematic drawing of the experimental setup for the 2-cell-population assay to allow ErbB4 activation exclusively at the cell membrane. The ErbB4 receptor (dark green rectangle) and the adapters (bright green circle) were transfected into population 1. The ligand neuregulin (Nrg) was transfected into population 2. This verifies that the Nrg induced ErbB4 activation and the subsequent phosphorylation-dependent interaction with the adapter protein are initiated via intercellular signaling at the cell membrane. (c) Visualisation of the 2-population assay. EYFP was co-transfected into population 1, ECFP into population 2. EYFP and ECFP were allowed to express for 20 h separately. Subsequently, both populations were mixed, and the assay was continued for additional 24 h. The merged image shows that YFP and CFP signals are not overlapping and that yellow and cyan signals are frequently in close proximity. Images were taken immediately before the luciferase assay was performed. (d) 2-population luciferase assay depicting the activation of the neuregulin-1 (Nrg1-II-b1a variant) induced phosphorylation-dependent interaction between the ErbB4 receptor and the adapter proteins PI3Kp85α, PI3Kp85β, Grb2 and Shc1. Combination of constructs were transfected into PC12 cells as indicated. RLUs, relative luciferase units; n = 6. (e) Renilla luciferase readings taken from the same assay as depicted in (d). Stimulation with Nrg1-II-b1a does not lead to substantial differences in absolute Renilla luciferase readings.