Schematic representation of the MAPPIT and relay MAPPIT principle. (A) JAK/STAT pathway. Stimulation of the leptin receptor (LR) by its ligand leads to activation of the associated JAK2 kinases and subsequent phosphorylation of the tyrosines of the LR by the JAKs. Several proteins including STAT3 are recruited to these phospho-tyrosine motifs leading to phosphorylation of STAT3 and its migration to the nucleus, where STAT3 will induce specific gene transcription. (B) Principle of MAPPIT. Ligand-induced activation of the leptin receptor-associated JAK kinases puts the receptor complex in a “stand-by” mode without induction of detectable reporter activity. No STAT3 recruitment and activation can occur due to the Y1138F mutation in the cytosolic domain of the leptin receptor. The only possible tyrosine phosphorylation site on the chimeric receptor is the C-terminal bait, which contains a part of the intracellular domain of the EpoR. Complementation is induced on a cognate bait-prey interaction, which leads to recruitment of the C-terminal part of gp130, containing 4 functional STAT3 recruitment sites. Subsequent ligand-dependent STAT3 phosphorylation and activation induces luciferase activity under control of the rPAP1 promoter. Hinge regions preceding the prey and bait provide additional flexibility in the chimeric polypeptides. (C) Principle of relay MAPPIT. Because the EpoR is a STAT5-dependent receptor, STAT3 activation can only occur via a cognate bait-prey interaction and recruitment via the STAT3-binding sites of the gp130 chain of the prey. Read-out is again based on the induction of luciferase activity under control of the rPAP1 promoter.