Cell and Molecular Biology Program, Colorado State University, Fort Collins, Colorado 80523, USA.
Dalton Trans. 2012 Jun 7;41(21):6419-30. doi: 10.1039/c2dt30521f. Epub 2012 Apr 30.
The effects of treatment with bis(maltolato)oxovanadium(IV) (BMOV) on protein localization in membrane microdomains were investigated by comparing the effects of insulin and treatment with BMOV on the lateral motions and compartmentalization of individual insulin receptors (IR). In addition, effects of insulin and BMOV on the association of IR, phosphorylated IR (pIR) and phosphorylated insulin receptor substrate-1 (pIRS-1) with chemically-isolated plasma membrane microdomains on rat basophilic leukemia (RBL-2H3) cells were evaluated. Single particle tracking experiments indicate that individual quantum dot-labeled IR on RBL-2H3 cells exhibit relatively unrestricted lateral diffusion of approximately 1 × 10(-10) cm(2) s(-1) and are confined in approximately 475 nm diameter cell-surface membrane compartments. After treating of RBL-2H3 cells with 10 μM BMOV, IR lateral diffusion and the size of IR-containing membrane compartments is significantly reduced to 6 × 10(-11) cm(2) s(-1) and approximately 400 nm, respectively. BMOV treatment also increases the association of IR with low-density, detergent-resistant membrane fragments isolated using isopycnic sucrose-gradient centrifugation from 2.4% for untreated cells to 25.8% for cells treated with 10 μM BMOV. Additionally, confocal fluorescence microscopic imaging of live RBL-2H3 cells labeled with the phase sensitive aminonaphthylethenylpyridinium-based dye, Di-4-ANEPPDHQ, indicates that BMOV treatment, but not insulin treatment, decreases cell-surface plasma membrane lipid order while fluorescence correlation spectroscopy measurements suggest that BMOV treatment does not affect IR surface-density or insulin binding affinity. Finally, model studies using microemulsions of cetyltrimethylammonium bromide (CTAB) micelles and (1)H NMR spectroscopy show that an oxidized form of BMOV readily localizes near the CTAB head-groups at the lipid-water interface. These observations were supported by IR spectroscopic studies using microemulsions of CTAB reverse micelles showing that both BMOV and oxidized BMOV are associated with the water pool. This conclusion is based on changes in (1)H NMR chemical shifts observed for the complex, oxidized BMOV. Moreover, these shifts appeared to be informative about the location of the complex. No differences were observed in the OD absorption peak positions for the CTAB reverse micelles prepared in the presence and absence of BMOV, oxidized BMOV or maltol. Combined, these results suggest that activation of IR signaling by both insulin and BMOV treatment involves increased association of IR with specialized, nanoscale membrane microdomains. The observed insulin-like activity of BMOV or decomposition products of BMOV may be due to changes in cell-surface membrane lipid order rather than due to direct interactions with IR.
研究了双（麦芽酚）氧钒（IV）（BMOV）治疗对膜微区中蛋白质定位的影响，方法是比较胰岛素和 BMOV 治疗对单个胰岛素受体（IR）的横向运动和区室化的影响。此外，还评估了胰岛素和 BMOV 对 IR、磷酸化 IR（pIR）和磷酸化胰岛素受体底物-1（pIRS-1）与用化学方法分离的大鼠嗜碱性白血病（RBL-2H3）细胞质膜微区的结合的影响。单颗粒跟踪实验表明，RBL-2H3 细胞上的单个量子点标记的 IR 表现出相对不受限制的约 1×10（-10）cm2s（-1）的横向扩散，并局限于约 475nm 直径的细胞表面膜区室中。用 10μMBMOV 处理 RBL-2H3 细胞后，IR 的横向扩散和包含 IR 的膜区室的大小分别显著降低至 6×10（-11）cm2s（-1）和约 400nm。BMOV 处理还增加了从未处理的细胞的 2.4%到用 10μMBMOV 处理的细胞的 25.8%的低密度、去垢剂抗性膜片段与 IR 的结合。此外，用基于对氨基萘基乙烯吡啶的相敏性染料 Di-4-ANEPPDHQ 标记活 RBL-2H3 细胞的共焦荧光显微镜成像表明，BMOV 处理而不是胰岛素处理降低了细胞表面质膜脂质有序性，而荧光相关光谱测量表明 BMOV 处理不影响 IR 表面密度或胰岛素结合亲和力。最后，使用十六烷基三甲基溴化铵（CTAB）胶束的微乳液和（1）H NMR 光谱的模型研究表明，BMOV 的氧化形式容易在脂质-水界面处定位于 CTAB 头基附近。IR 光谱研究使用 CTAB 反胶束微乳液支持了这些观察结果，表明 BMOV 和氧化 BMOV 都与水相有关。这一结论基于观察到复合物，氧化 BMOV 的（1）H NMR 化学位移的变化。此外，这些位移似乎与复合物的位置有关。在存在和不存在 BMOV、氧化 BMOV 或麦芽酚的情况下制备的 CTAB 反胶束的 OD 吸收峰位置没有观察到差异。综上所述，胰岛素和 BMOV 处理激活 IR 信号都涉及到 IR 与专门的纳米尺度膜微区的关联增加。BMOV 或 BMOV 分解产物的胰岛素样活性可能是由于细胞表面膜脂质有序性的变化，而不是由于与 IR 的直接相互作用。
