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J. Biol. Chem., Vol. 283, Issue 18, 12643-12653, May 2, 2008

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Unexpected Complexity in the Mechanisms That Target Assembly of the Spectrin Cytoskeleton^*

Amlan Das, Christine Base, Debasis Manna, Wonhwa Cho, and Ronald R. Dubreuil¹

From the Laboratory for Molecular Biology and Department of Biological Sciences and the Department of Chemistry, University of Illinois, Chicago, Illinois 60607

The spectrin cytoskeleton assembles within discrete regions of the plasma membrane in a wide range of animal cell types. Although recent studies carried out in vertebrate systems indicate that spectrin assembly occurs indirectly through the adapter protein ankyrin, recent studies in Drosophila have established that spectrin can also assemble through a direct ankyrin-independent mechanism. Here we tested specific regions of the spectrin molecule for a role in polarized assembly and function. First, we tested mutant β-spectrins lacking ankyrin binding activity and/or the COOH-terminal pleckstrin homology (PH) domain for their assembly competence in midgut, salivary gland, and larval brain. Remarkably, three different assembly mechanisms operate in these three cell types: 1) neither site was required for assembly in salivary gland; 2) only the PH domain was required in midgut copper cells; and 3) either one of the two sites was sufficient for spectrin assembly in larval brain. Further characterization of the PH domain revealed that it binds strongly to lipid mixtures containing phosphatidylinositol 4,5-bisphosphate (PIP₂) but not phosphatidylinositol 3,4,5-trisphosphate. A K8Q mutation in the lipid binding region of the PH domain eliminated the PIP₂ interaction in vitro, yet the mutant protein retained full biological function in vivo. Reporter gene studies revealed that PIP₂ and the spectrin PH domain codistribute with one another in cells but not with authentic wild type β-spectrin. Thus, it appears that the PH domain imparts membrane targeting activity through a second mechanism that takes precedence over its PIP₂ binding activity.

Received for publication, January 4, 2008

^* This work was supported by National Institutes of Health Grants GM49301 (to R. R. D.) and GM68849 (to W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 900 S. Ashland Ave., Rm. 4152 MBRB (m/c567), Chicago, IL 60607. Fax: 312-413-2691; E-mail: ron{at}uic.edu.

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