title: The IA-2 interactome.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Experimental Medicine Section, Oral Infection and Immunity Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Y F
authors.initials: YF
authors.lastname: Hu
authors.forename: H L
authors.initials: HL
authors.lastname: Zhang
authors.forename: T
authors.initials: T
authors.lastname: Cai
authors.forename: S
authors.initials: S
authors.lastname: Harashima
authors.forename: A L
authors.initials: AL
authors.lastname: Notkins
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015551:Autoimmunity / Q000276:immunology; D002460:Cell Line; D021984:Cyclophilin A / Q000378:metabolism; D053418:Death Domain Receptor Signaling Adaptor Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D022142:Secretory Vesicles / Q000737:chemistry*; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D001324:Autoantigens; D053418:Death Domain Receptor Signaling Adaptor Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; C105852:MADD protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D021984:Cyclophilin A