Secretory granules (SGs) of endocrine cells are responsible for storage and regulated secretion of peptide hormones, including insulin. Pharmacological studies suggest that both tyrosine phosphorylation and dephosphorylation pathways modulate the trafficking of SGs, but the molecules involved in these pathways have not yet been identified. Islet Cell Autoantigen (ICA) 512 is a novel member of the receptor protein tyrosine phosphatase (RPTP) family that we have shown to be an intrinsic membrane protein of SGs. Interestingly, ICA512 contains an atypical protein tyrosine phosphatase (PTP) domain and does not display enzymatic activity. In view of its localization and homology with RPTPs, we hypothesize that ICA512 represents a link between regulated secretion and signal transduction pathways mediated via tyrosine phosphorylation. We plan to test this hypothesis by establishing the protein's function. To this aim we propose first to identify ICA512 interacting proteins using three complementary approaches: 1) the two- hybrid system in yeast, 2) co-purification by solid phase affinity chromatography and immunoprecipitation with anti-ICA512 antibodies, and 3) affinity purification on recombinant ICA512. ICA512 interactors will be cloned and characterized for their affinity to wild-type and ICA512 mutants. Knowledge of ICA512 interactors will provide clues about the cell pathways to which ICA512 insulinoma cell clones overexpressing wild-type or mutant ICA512 alleles in a tetracycline-inducible fashion. The role of ICA512 in vivo will be tested by conditionally over- expressing wild-type or a PTP active mutant of ICA512 in the pancreatic beta-cells of transgenic mice. This will be accomplished using the reverse tetracycline-inducible expression system driven by the rat insulin promoter II. As RPTPs are known to play an important developmental role, we will test the potential impact of ICA512 transgenes on islet organogenesis and beta-cell differentiation by inducing their overexpression at different stages of mouse development. The potential role of ICA512 in beta-cell physiology will be established by monitoring glucose homeostatis in transgenic mice and by studying insulin secretion from purified transgenic islets. These studies could establish a direct relationship between regulated secretion and signal transduction via tyrosine phosphorylation. In addition, since atypical PTP domains similar to that of ICA512 are present in an increasing number of orphan genes, our studies on ICA512 may provide insight into the general significance of such protein motifs.
