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J. Biol. Chem., Vol. 283, Issue 15, 9623-9632, April 11, 2008

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Identification of Alix-type and Non-Alix-type ALG-2-binding Sites in Human Phospholipid Scramblase 3

DIFFERENTIAL BINDING TO AN ALTERNATIVELY SPLICED ISOFORM AND AMINO ACID-SUBSTITUTED MUTANTS^*

From the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca²⁺-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2^GF122, pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2^GF122. Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca²⁺-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2^GF122. The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe⁴⁹ was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2^GF122: (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.

Received for publication, January 28, 2008

^* This work was supported by a Grant-in-Aid for Scientific Research (B) (to M. M.) and a Grant-in-Aid for Young Scientists (B) (to H. S.). 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 text, references, Table S1, and Figs. S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 81-52-789-4088; Fax: 81-52-789-5542; E-mail: mmaki{at}agr.nagoya-u.ac.jp.

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