Comparative analysis of the C-terminal membrane association domains of b(Heavy)-spectrin and b-spectrin. B. MacIver ^{1, 2}, G.H. Thomas ^{1, 2}. 1) Dept Biol,; 2) Dept Biochem. and Molec. Biol., The Pennsylvania State University., University Park, PA.

   Drosophila has two spectrin isoforms; (ab)₂ and (ab_H)₂ derived from three genes; a-spectrin, b-spectrin and karst for b(Heavy)-spectrin. Mutations in the a-spectrin gene cause early stage larval lethality whereas b-spectrin mutations result in late embryonic lethality. karst mutations are mostly lethal with adult escapers showing defects in tissues of epithelial origin including the eye, wing and trachea. Both b_H- and b-spectrin have C-termini containing a pleckstrin homology (PH) domain, but are divergent in the surrounding sequences. The b_H- C-terminus is much larger (501aa vs. 201aa) and includes a polyglutamine (OPA) repeat.
   Dominant-negative expression of the b_H- C-terminus in the eye after onset of differentiation disrupts normal development resulting in ommatidial fusions, loss of photoreceptor morphology and loss of pigmentation, mostly via apoptosis. Expression of the b-spectrin C-terminus does not cause this effect and deletion derivatives of b_H- implicate the PH domain and 3' regions immediately C-terminal in this effect.
    PH domains are known to bind phosphoinositides and there is accumulating evidence that they also interact with other proteins. We have expressed both b- and b_H- domains as bacterial fusion proteins and are in the process of evaluating the phosphoinositide binding capabilities of each construct. We are also seeking interacting loci by conducting a screen using the dominant-negative phenotype. We will present our latest results using these approaches.