IDENTIFICATION OF THE BINDING PARTNERS FOR FLIGHTLESS-I, A NOVEL PROTEIN BRIDGING THE LEUCINE-RICH REPEAT AND THE GELSOLIN SUPERFAMILIES

Flightless-I (fliI) is a novel member of the gelsolin family that is i mportant for actin organization during Drosophila embryogenesis and my ogenesis. Drosophila fliI and the human homolog FLI both contain the c lassic gelsolin 6-fold segmental repeats and an amino-terminal extensi on of 16 tandem leucine-rich repeats (LRR). LRR repeats form amphipath ic beta-alpha structural units that mediate protein-protein interactio ns. Although there are close to 100 known LRR domain-containing protei ns, only a few binding pairs have been identified. In this paper, we u sed biochemical and genetic approaches to identity proteins that inter act with human FLI. In vitro synthesized FLI bound to actin-Sepharose and binding was reduced by competition with excess soluble actin. Acti n binding was mediated though the gelsolin-like domain and not the LRR domain. Although the FLI LRR module is most closely related to the LR R domains of Ras-interactive proteins, FLI does not associate with Ras , selected Ras effectors, or other Ras-related small GTPases. Two-hybr id screens using FLI LRR as bait identified a novel LRR binding partne r. The 0.65-kilobase pair (kb) clone from the screen survived addition al rounds of stringent two-hybrid pairwise assays, establishing a spec ific interaction. Binding FLI LRR was corroborated by co-immunoprecipi tation with FLI LRR. The translated sequence of the FLI LRR associated protein (FLAP) encodes a novel protein not represented in the data ba se. Northern blot analyses revealed four FLAP messages of approximatel y 2.7, 2.9, 3.3, and 5.1 kb, which are differentially expressed in the tissues tested. Skeletal and cardiac muscles are particularly rich in the 3.3-kb FLAP message, and the FLI message aas well. Full-length FL AP clones were isolated from a mouse skeletal muscle cDNA library. The y have an open reading frame which encodes for a protein containing 62 6 amino acids. Sequence analyses predict that the FLAP protein is rich in alpha-helices and contains stretches of dimeric coiled coil in its middle region and COOH terminus. The identification of actin and FLAP as a binding ligands for the gelsolin-like domain and the LRR domain, respectively, suggests that FLI may link the actin cytoskeleton to ot her modules implicated in intermolecular recognition and structural or ganization.