The interactions among upd-family ligands. Qian Chen, Douglas Harrison. Dept Biol, Univ Kentucky, Lexington, KY.

   The JAK/STAT signaling pathway is the major signaling cascade in response to a variety of cytokines and growth factors in vertebrates and it is highly conserved. But unlike vertebrates, the Drosophila JAK/STAT signaling pathway has only three identified ligands: Unpaired(Upd), Upd2 and Upd3. The expression patterns of upd2 and upd3 overlap with that of upd during several developmental processes. upd2 and upd are expressed in identical stripes in embryos, while upd3 and upd are co-expressed in the polar cells of egg chambers and posterior region of eye discs. Given the overlapping expression pattern, we hypothesize that the three ligands cooperatively regulate the JAK signaling by forming different ligand complexes. We tested the physical interaction among the three ligands by Bimolecular Fluorescence Complementation (BiFC). All three ligands show the ability to form homodimers, and the interaction of Upd2 and Upd3 homodimers were stronger than Upd homodimers. Upd2 and Upd3 were able to form heterodimers with Upd individually as well. Homotypic interaction between Upd3 molecules was also detected in a yeast two-hybrid assay. To determine the putative functional domains of the upd-family ligands, we compared the sequence of the three ligands and identified six short conserved domains. We substituted each of the conserved domains on upd3 with five alanine residues individually and tested their function in a luciferase reporter assay. All six alanine substitutions dramatically reduced the Upd3 ability in stimulating JAK signaling. Interactions between the six alanine substituted upd3 molecules with intact ligands will be tested in BiFC assay to see if any of them are responsible for ligand interactions.