The IA-2 interactome.
Islet antigen-2 (IA-2), a major autoantigen in type 1 diabetes, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase (PTP) family. IA-2 is located in dense-core secretory vesicles and is involved in the regulation of insulin secretion. The present experiments were initiated to identify those proteins that interact ... with IA-2 (i.e. the IA-2 interactome) as a first step towards elucidating the mechanism(s) by which IA-2 influences insulin secretion and serves as an autoantigen.To determine the proteins with which IA-2 interacts, a yeast two-hybrid system was used to screen a human foetal library, and deletion mutants were used to determine the binding sites. Positive interactions were confirmed by immunoprecipitation pull-down experiments using cell lysate from transfected mammalian cell lines.Six new interacting proteins were identified by this approach: mitogen-activated protein kinase-activating death domain (MADD), the MADD isoform IG20, PTPrho, PTPsigma, sorting nexin 19 (SNX19) and cyclophilin A. Using a series of IA-2 deletion mutants, we identified the regions on the IA-2 molecule to which five of the interacting proteins bound. Amino acids 744-979 of IA-2 were required for the maximum binding of MADD, IG20 and SNX19, whereas amino acids 602-907 of IA-2 were required for the maximum binding of PTPrho and PTPsigma. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2.The IA-2 interactome based on, pull-down experiments, currently consists of 12 proteins. The identification of these interacting proteins provides clues as to how IA-2 exerts its biological functions.
Mesh Terms:
Animals, Autoantigens, Autoimmunity, Cell Line, Cyclophilin A, Death Domain Receptor Signaling Adaptor Proteins, Guanine Nucleotide Exchange Factors, Humans, Immunoprecipitation, Insulin, Insulin Secretion, Membrane Proteins, Mutation, Protein Binding, Protein Interaction Mapping, Protein Isoforms, Protein Tyrosine Phosphatase, Non-Receptor Type 1, Protein Tyrosine Phosphatases, Receptor-Like Protein Tyrosine Phosphatases, Class 8, Secretory Vesicles, Transfection, Two-Hybrid System Techniques
Animals, Autoantigens, Autoimmunity, Cell Line, Cyclophilin A, Death Domain Receptor Signaling Adaptor Proteins, Guanine Nucleotide Exchange Factors, Humans, Immunoprecipitation, Insulin, Insulin Secretion, Membrane Proteins, Mutation, Protein Binding, Protein Interaction Mapping, Protein Isoforms, Protein Tyrosine Phosphatase, Non-Receptor Type 1, Protein Tyrosine Phosphatases, Receptor-Like Protein Tyrosine Phosphatases, Class 8, Secretory Vesicles, Transfection, Two-Hybrid System Techniques
Diabetologia
Date: Dec. 01, 2005
PubMed ID: 16273344
View in: Pubmed Google Scholar
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