Aims/hypothesis  

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.

Materials and 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, PTPρ, PTPσ, 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 PTPρ and PTPσ. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2.

Conclusions/interpretation  

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.