Valverde A M, Lorenzo M, Pons S, White M F, Benito M
Departamento de Bioquimica y Biologia Molecular II, Centro Mixto Consejo Superior de Investigaciones Cientificas/Universidad Complutense, Facultad de Farmacia, Madrid, Spain.
Mol Endocrinol. 1998 May;12(5):688-97. doi: 10.1210/mend.12.5.0106.
In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.
在本研究中，我们研究了胰岛素受体底物蛋白（IRS-1和IRS-2）对胎鼠棕色脂肪细胞中胰岛素/胰岛素样生长因子I（IGF-I）信号通路的作用，该模型同时表达胰岛素和IGF-I受体。胰岛素/IGF-I迅速刺激IRS-1和IRS-2的酪氨酸磷酸化、它们与p85α的结合以及与IRS-1和IRS-2相关的磷脂酰肌醇（PI）3激酶的激活，且程度相同，在相同生理剂量（10 nM）下，胰岛素的作用强于IGF-I。此外，胰岛素/IGF-I刺激了与IRS-1相关的Grb-2磷酸化。然而，几乎未检测到与IRS-2相关的Grb-2磷酸化。用含有p85α SH2结构域的谷胱甘肽-S-转移酶融合蛋白进行的下拉实验表明，响应胰岛素/IGF-I时，IRS-1和IRS-2与p85α有很强的结合，胰岛素的作用强于IGF-I。然而，Grb-2-SH2结构域显示出功能差异。虽然发现IRS-1/Grb-2之间有很强
