PC-1 Insulin Receptor Signaling

描述(申请人提供)：胰岛素抵抗是2型糖尿病(T2D)和胰岛素抵抗综合征患者的一个特征。PC-1是一种II类质膜外蛋白，它抑制位于残基485-599之间的IRα亚基。这个IR区域将阿尔法亚单位配体结合域与β亚单位酪氨酸激酶结构域连接起来。在大多数有胰岛素抵抗的受试者中，我们和其他人发现肌肉和其他组织中的PC-1要么过度表达，要么处于更活跃的形式(Q等位基因)。PC-1基因的转染和过表达可选择性地降低IR酪氨酸激酶活性和IR信号。我们现在发现，人类PC-1在小鼠肌肉和肝脏中的过度表达在体内会导致胰岛素抵抗和糖尿病。因此，我们假设PC-1是胰岛素抵抗的主要原因。在这里，我们计划证明PC-1是胰岛素作用的重要调节因子，确定PC-1如何与IR相互作用，并采用体外和体内的策略来拮抗PC-1。我们提出如下建议：首先，我们计划对过度表达PC-1各种等位基因的小鼠进行代谢表型表征。我们将使用腺病毒介导的PC-1在肝脏过度表达的小鼠，以及普遍和组织特异性PC-1过度表达的转基因小鼠。其次，利用我们的PC-1过表达动物模型，我们将研究抗PC-1单抗、PC-1RNAi和PC-1反义寡聚体是否会降低PC-1水平并改善胰岛素的作用。为了调节PC-1水平，我们还将使用Tet Off/On系统。第三，由于我们在体外和体内的数据表明PC-1直接与IRα亚基相互作用，我们将通过阐明PC-1如何以及在哪里与IR结合来研究PC-1与IR的相互作用。为此，我们将采用直接约束性研究。此外，还将产生IR和PC-1的突变体来定位蛋白质-蛋白质相互作用的离散位点。通过定义PC-1和IR之间的接触点，我们有可能设计出抑制这种相互作用的策略。第四，培养成纤维细胞中PC-1含量与肌肉活检密切相关。因此，利用胰岛素抵抗患者的成纤维细胞，将探索导致胰岛素抵抗患者PC-1过度表达的机制。因此，我们将确定PC-1在成纤维细胞中的过度表达是否由转录和/或转录后机制引起。

DESCRIPTION (provided by applicant): Resistance to insulin is a feature of patients with type 2 diabetes mellitus (T2D) and the insulin resistance syndrome. PC-1, a class II plasma membrane exoprotein inhibits the IR alpha subunit in a region between residues 485-599. This IR region links the alpha subunit ligand binding domain to the beta subunit tyrosine kinase domain. In most subjects with insulin resistance, we and others have found PC-1 in muscle and other tissues is either over expressed or is in a more active form (Q allele). Transfection and overexpression of PC-1 into cultured cells selectively reduces both IR tyrosine kinase activity and IR signaling. We now find that human PC-1 overexpression in mouse muscle and liver causes in vivo insulin resistance and diabetes. We hypothesize, therefore, that PC-1 is a major cause of insulin resistance. Herein we plan to document that PC-1 is an important regulator of insulin action, define how PC-1 interacts with the IR, and employ strategies both in vitro and in vivo to antagonize PC-1. We propose the following: First, we plan to metabolically phenotypically characterize mice that are over expressing the various alleles of PC-1. We will employ mice with adenovirus-mediated PC-1 overexpression in liver, and transgenic mice with general and tissue-specific PC-1 overexpression. Second, employing our animal models of PC-1 overexpression, we will investigate whether anti PC-1 monoclonal antibodies, PC-1 RNAi, and PC-1 antisense oligomers will lower PC-1 levels and improve insulin action. To regulate PC-1 levels, we will also use the Tet off/on system. Third, because we have data both in vitro and in vivo indicating that PC-1 directly interacts with the IR alpha subunit, we will investigate the interactions of PC-1 with the IR by elucidating how and where PC-1 binds to the IR. For this purpose, we will employ direct binding studies. In addition, mutants of both the IR and PC-1 will be produced to locate discrete sites of protein-protein interaction. By defining the contact points between PC-1 and the IR, we have the potential to devise strategies to inhibit this interaction. Fourth, PC-1 content in cultured fibroblasts and muscle biopsy closely correlate. Therefore, employing fibroblasts from insulin resistant patients, the mechanisms that cause PC-1 overexpression in insulin resistant humans will be explored. We will determine therefore whether PC-1 overexpression in fibroblast is caused by transcriptional and/or post-transcriptional mechanisms.