MaCaulay S Lance, Stoichevska Violet, Grusovin Julian, Gough Keith H, Castelli Laura A, Ward Colin W
CSIRO Health Sciences and Nutrition, 343 Royal Pde., Parkville, Victoria 3052, Australia.
Biochem J. 2003 Nov 15;376(Pt 1):123-34. doi: 10.1042/BJ20030130.
SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.
分选连接蛋白9（SNX9）是参与蛋白质运输的蛋白家族中的一员。该家族的特征是具有一个独特的PX（Phox同源）结构域，其中包括富含脯氨酸的序列和上游磷脂结合结构域。许多分选连接蛋白，包括SNX9，还具有C端卷曲区域。SNX9另外还有一个N端SH3（Src同源3）结构域。在此，我们研究了SNX9的细胞定位及其在胰岛素作用中可能发挥的作用。在3T3L1脂肪细胞中，SNX9呈胞质点状分布，与内体和胞质定位一致，且被排除在细胞核之外。SH3结构域至少部分负责SNX9的膜定位，因为在HEK293T细胞中表达缺失SH3结构域的绿色荧光蛋白（GFP）-SNX9融合蛋白会使该蛋白定位于胞质中。膜定位也可能部分归因于PX结构域，因为体外磷脂结合研究表明SNX9与多磷酸肌醇结合。胰岛素诱导SNX9从胞质向膜部分移动。体外实验中，谷胱甘肽S-转移酶（GST）-SNX9融合蛋白与胰岛素样生长因子1（IGF1）及胰岛素受体相关。在3T3L1脂肪细胞中过表达的GFP-SNX9融合蛋白与胰岛素受体进行了共免疫沉淀。此外，在CHO-T细胞中过表达这种GFP-SNX9融合蛋白会降低胰岛素结合，这与SNX9在胰岛素受体运输中的作用一致。用抗SNX9抗体显微注射3T3L1细胞可抑制胰岛素对葡萄糖转运蛋白4（GLUT4）转位的刺激作用。这些结果支持SNX9通过影响胰岛素受体的加工/运输参与胰岛素作用。同时也提示其在调节GLUT4的细胞加工、运输和/或亚细胞定位方面具有次要作用。
