The IA-2 interactome

• Published in: Diabetologia 2005-12, Vol.48 (12), p.2576-2581
• Main Authors: HU, Y. F, ZHANG, H. L, CAI, T, HARASHIMA, S, NOTKINS, A. L
• Format: Article
• Language: English
• Subjects: Animals; Autoantigens - analysis; Autoantigens - genetics; Autoantigens - immunology; Autoantigens - metabolism; Autoimmunity - immunology; Biological and medical sciences; Cell Line; Cyclophilin A - metabolism; Death Domain Receptor Signaling Adaptor Proteins; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Guanine Nucleotide Exchange Factors - metabolism; Humans; Immunoprecipitation; Insulin - metabolism; Insulin Secretion; Medical sciences; Membrane Proteins - analysis; Membrane Proteins - genetics; Membrane Proteins - immunology; Membrane Proteins - metabolism; Mutation; Protein Binding; Protein Interaction Mapping; Protein Isoforms; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases - analysis; Protein Tyrosine Phosphatases - genetics; Protein Tyrosine Phosphatases - immunology; Protein Tyrosine Phosphatases - metabolism; Receptor-Like Protein Tyrosine Phosphatases, Class 8; Secretory Vesicles - chemistry; Transfection; Two-Hybrid System Techniques
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s00125-005-0037-y

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.