mappit_concept

MAPPIT concept

Upon ligand binding, class I cytokine receptor complexes are reorganized, leading to the cross-activation of associated Janus kinases (JAKs). These kinases phosphorylate specific tyrosine residues along the receptor tails, turning these into docking sites for signalling molecules including members of the Signal Transducer and Activator of Transcription (STAT) family. STATs are in turn activated by phosphorylation and migrate to the nucleus, where they induce specific target gene transcription.

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The MAPPIT strategy takes advantage of the fact that activated JAKs are able to phosphorylate STAT recruitment sites in trans. The bait protein is fused to a mutant receptor that still allows JAK activation, but from which STAT target sites have been eliminated (hybrid one). The prey protein is tethered to a receptor fragment containing functional STAT recruitment sites (hybrid two). Bait-prey interaction leads to functional complementation of the signalling pathway, whereby phosphorylation of the prey chimeras leads to recruitment and activation of STAT molecules.

In the original method, the bait receptor chimera consists of the extracellular and transmembrane part of the homodimeric Epo receptor fused to the intracellular portion of the leptin receptor in which three tyrosine residues have been mutated to phenylalanine. The prey is fused to a fragment of the gp130 receptor chain, which harbours several STAT3 recruitment sites. Read-out is either a STAT3 phosphorylation test or a STAT3-based reporter assay leading to the expression of luciferase or an IL5Rα-derived membrane-tag.

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