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.
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 Ca(2+)-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(DeltaGF122), 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(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) 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(DeltaGF122): (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.
Mesh Terms:
Alternative Splicing, Amino Acid Substitution, Annexin A7, Annexins, Apoptosis Regulatory Proteins, Binding Sites, Calcium-Binding Proteins, Carrier Proteins, Cell Cycle Proteins, Cell Line, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Phospholipid Transfer Proteins, Protein Binding, Protein Isoforms, Recombinant Fusion Proteins, Transcription Factors, Vesicular Transport Proteins
Alternative Splicing, Amino Acid Substitution, Annexin A7, Annexins, Apoptosis Regulatory Proteins, Binding Sites, Calcium-Binding Proteins, Carrier Proteins, Cell Cycle Proteins, Cell Line, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Phospholipid Transfer Proteins, Protein Binding, Protein Isoforms, Recombinant Fusion Proteins, Transcription Factors, Vesicular Transport Proteins
J. Biol. Chem.
Date: Apr. 11, 2008
PubMed ID: 18256029
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