MOLECULAR AND FUNCTIONAL ANALYSIS OF ICA 512

内分泌细胞的分泌颗粒(SGS)负责储存 以及调节包括胰岛素在内的多肽激素的分泌。 药理学研究表明，酪氨酸磷酸化和 去磷酸化途径调节SGS的运输，但 参与这些途径的分子还没有被确定。 胰岛细胞自身抗原(ICA)512是受体的新成员 蛋白酪氨酸磷酸酶(RPTP)家族，我们已经证明是一个 SGS的固有膜蛋白。有趣的是，ICA512包含一个 非典型蛋白酪氨酸磷酸酶(PTP)结构域，不显示 酶活性。鉴于它的本地化和同源性 RPTPS，我们假设ICA512代表受调控的 酪氨酸介导的分泌和信号转导途径 磷酸化。我们计划通过建立 蛋白质的功能。为此，我们建议首先将ICA512 使用三种互补方法相互作用的蛋白质：1)两种- 酵母杂交系统；2)固相亲和共纯化 用抗ICA512抗体进行层析和免疫沉淀，以及 3)重组ICA512的亲和纯化。ICA512交互组件将 克隆并鉴定其与野生型和ICA512的亲和力 变种人。关于ICA512交互作用的知识将提供关于 ICA512胰岛素瘤细胞克隆过表达的细胞途径 四环素可诱导的野生型或突变型ICA512等位基因。 ICA512在体内的作用将通过有条件的过度- ICA512野生型或PTP活性突变体在胰腺中的表达 转基因小鼠的β细胞。这将通过使用 大鼠驱动的四环素反向诱导表达系统 胰岛素促进剂II.已知的RPTPs在 发展作用，我们将测试ICA512的潜在影响 转基因对胰岛器官发生和胰岛β细胞分化的影响 诱导它们在小鼠发育的不同阶段过度表达。 ICA512在β细胞生理学中的潜在作用将被确定 通过监测转基因小鼠的葡萄糖稳态，并通过研究 纯化的转基因胰岛的胰岛素分泌。这些研究可能 在调节分泌和信号之间建立直接关系 通过酪氨酸磷酸化的转导。此外，由于非典型 与ICA512类似的PTP结构域在不断增加 关于孤儿基因的数量，我们对ICA512的研究可能会提供对 这些蛋白质基序的一般意义。

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.