Absence of functional insulin receptor substrate-3 (IRS-3) gene in humans
Absence of functional insulin receptor substrate-3 (IRS-3) gene in humans
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DOI：
10.1007/s00125-002-0945-z
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发表时间：
2002-12-01
期刊：
影响因子：
8.2
通讯作者：
Zierath, JR
中科院分区：
文献类型：
--
作者：
Björnholm, M;He, AR;Zierath, JR
目的/假设。胰岛素受体底物（IRS）蛋白在胰岛素作用和胰腺β细胞功能中起重要作用。至少有四种哺乳动物IRS分子已被确定。尽管编码人IRS-1、IRS-2和IRS-4的基因和cDNA已被克隆，但IRS-3仅在啮齿动物中被鉴定。因此，我们尝试克隆人IRS-3基因。对胰岛素刺激的大鼠或人脂肪细胞进行Western印迹分析以评估IRS-3酪氨酸磷酸化。筛选人肝脏和脂肪cDNA文库，试图克隆IRS-3 cDNA。设计了一种基于PCR的方法来扩增IRS-3 cDNA。以小鼠IRS-1、IRS-2和IRS-3基因为模板，采用逆转录PCR技术，对小鼠IRS-1、IRS-2和IRS-3基因进行计算机筛选，并对公开的人类基因组进行初步检索。在人类脂肪细胞中，我们没有检测到M-r 60 000磷蛋白对应的IRS-3，而在大鼠脂肪细胞IRS-3蛋白和胰岛素刺激的酪氨酸磷酸化很容易观察到。没有一种分子方法提供了在人体组织中存在功能性IRS-3基因的证据。应用生物信息学方法，在人IRS-3基因中发现两处缺失。预测人IRS-3基因产物缺乏磷酸酪氨酸结合结构域，也缺乏鼠IRS-3的相应氨基酸353-407的序列。这两个同源区域之间的基因组DNA连续序列不具有人IRS-3的编码信息。对人类IRS-3基因组区域的计算机筛选，结合进一步的生物学和分子验证，提供了针对人类IRS-3功能的证据。
Aim/hypothesis. Insulin receptor substrate (IRS) proteins play important roles in insulin action and pancreatic beta-cell function. At least four mammalian IRS molecules have been identified. Although genes and cDNAs encoding human IRS-1, IRS-2, and IRS-4 have been cloned, IRS-3 has been identified only in rodents. Thus, we have attempted to clone the human IRS-3 gene.Methods. Insulin-stimulated rat or human adipocytes were subjected to Western blot analysis to assess IRS-3 tyrosine phosphorylation. Human liver and adipose cDNA libraries were screened in an effort to clone IRS-3 cDNA. A PCR-based approach was designed to amplify IRS-3 cDNA. Reverse transcription PCR was carried out using mRNA from adipose tissue, liver, and skeletal muscle as templates in combination with an in silico screen using mouse IRS-1, IRS-2 and IRS-3 in a tblastn search of the draft public human genome.Results. In human adipocytes we did not detect a M-r 60 000 phosphoprotein corresponding to IRS-3, whereas in rat adipocytes IRS-3 protein and insulin-stimulated tyrosine phosphorylation was readily observed. None of the molecular approaches provided evidence for a functional IRS-3 gene in human tissue. Two deletions in human IRS-3 gene were identified using bioinformatics. The human IRS-3 gene product is predicted to lack a phosphotyrosine binding domain and also the sequence corresponding amino acid 353-407 of murine IRS-3. The contiguous sequence of genomic DNA between these two homologous regions does not have the coding information for human IRS-3.Conclusion/interpretation. In silico screening of the human IRS-3 genome region, combined with further biological and molecular validation, provides evidence against a functional IRS-3 in humans.