The third intracellular loop of Drosophila Lilipod is required for protein function in vivo and can mediate protein-protein interactions in vitro.

The evolutionarily conserved Lipocalin-Interacting Membrane Receptor (LIMR) family (InterPro: IPR006876) consists of transmembrane (TM) proteins characterized by 9 TM domains (TMDs). Their reported biological functions are diverse and remain poorly understood. In previous work, we showed that the fly family member Lilipod (Lili) impacts biological processes regulated by the fly BMP/TGF-β ligand Decapentaplegic (Dpp), including germline stem cell self-renewal in the Drosophila ovary, dorsal closure during embryonic development and wing vein formation at the pupal stage. Based on this genetic evidence, Lili directly or indirectly enhances bone morphogenetic protein (BMP) signaling. In the ovary, Lili functions between the activated type I BMP receptor and the SMAD intracellular transducer. To gain insight into Lili function at the cellular and molecular levels, we probed the functional significance of its largest intracellular loop, Intracellular Loop 3 (ICL3). Through mutational analysis, we mapped sequences critical for Lili function in vivo to the evolutionarily conserved regions of ICL3. Additionally, we showed that fly-human chimeric proteins in which Lili ICL3 is replaced with the ICL3 of its human homologs, LMBR1 and LMBR1L, can rescue lili null-mutant phenotypes. Using ICL3 as bait in an unbiased Yeast 2-Hybrid (Y2H) screen, we identified putative interactors, including the BMP signaling cascade components Mad, Sara, Nup93 and Nup358, and further Y2H analyses identified distinct regions on ICL3 as potentially important for protein binding. Taken together, our work has identified ICL3 as a region that is critical for Lili protein function, most likely via its mediation of protein-protein interactions (PPIs).