PC-1 Insulin Receptor Signaling

描述（由申请方提供）：胰岛素抵抗是2型糖尿病（T2 D）和胰岛素抵抗综合征患者的一个特征。PC-1，一种II类质膜外蛋白，在残基485-599之间的区域抑制IR α亚基。该IR区域将α亚基配体结合结构域连接至β亚基酪氨酸激酶结构域。在大多数胰岛素抵抗受试者中，我们和其他人发现PC-1在肌肉和其他组织中过度表达或处于更活跃的形式（Q等位基因）。PC-1转染和过表达到培养细胞中选择性地降低IR酪氨酸激酶活性和IR信号传导。我们现在发现人PC-1在小鼠肌肉和肝脏中的过表达导致体内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。在此，我们计划证明PC-1是胰岛素作用的重要调节剂，定义PC-1如何与IR相互作用，并采用体外和体内拮抗PC-1的策略。我们提出以下建议：首先，我们计划代谢表型表征小鼠过度表达PC-1的各种等位基因。我们将采用腺病毒介导的PC-1在肝脏中过表达的小鼠，以及具有一般和组织特异性PC-1过表达的转基因小鼠。第二，利用我们的PC-1过表达的动物模型，我们将研究抗PC-1单克隆抗体、PC-1 RNAi和PC-1反义寡聚体是否会降低PC-1水平并改善胰岛素作用。为了调节PC-1水平，我们还将使用泰特关/开系统。第三，因为我们有体外和体内的数据表明PC-1直接与IR α亚基相互作用，我们将通过阐明PC-1如何和在何处与IR结合来研究PC-1与IR的相互作用。此外，将产生IR和PC-1的突变体以定位蛋白质-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，培养的成纤维细胞中PC-1含量与肌肉活检密切相关。因此，采用来自胰岛素抵抗患者的成纤维细胞，将探索导致胰岛素抵抗人类中PC-1过表达的机制。因此，我们将确定PC-1在成纤维细胞中的过表达是否是由转录和/或转录后机制引起的。

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.