Issad T, Boute N, Boubekeur S, Lacasa D
Department of Cell Biology, Institut Cochin, CNRS-UMR 8104, Inserm U567, Université Paris V, 22, rue Méchain, 75014 Paris, France.
Biochimie. 2005 Jan;87(1):111-6. doi: 10.1016/j.biochi.2004.12.008.
PTP1B is a protein tyrosine-phosphatase predominantly located on the cystosolic surface of the endoplasmic reticulum. This tyrosine-phosphatase plays a major role in the regulation of the activity of the insulin receptor (IR). We have studied the interaction of the IR with PTP1B in living cells using bioluminescence resonance energy transfer (BRET). The IR was fused to Renilla luciferase and a substrate-trapping mutant of PTP1B was fused to the yellow variant of the green fluorescent protein (YFP). When the two partners interacted, an energy transfer occurred between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, could be detected. The interaction of the IR with PTP1B could be monitored in real time for more than 30 min. Insulin rapidly and dose-dependently stimulated this interaction. The basal (insulin-independent) interaction of IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B, indicating that this basal interaction mainly occurred in the endoplasmic reticulum. In the basal state, PTP1B and the IR indeed co-localized in the endoplasmic reticulum, as demonstrated by confocal microscopy and cell fractionation experiments. Moreover, inhibition of IR processing with tunicamycin indicated that the basal interaction of PTP1B with IR occurred during biosynthesis of the IR precursor in the endoplasmic reticulum. These results strongly suggest that PTP1B not only dephosphorylates the insulin receptor that has been activated by insulin, but also regulates the insulin receptor precursor during its biosynthesis. Localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the immature insulin receptor precursor.
蛋白酪氨酸磷酸酶1B（PTP1B）是一种主要位于内质网胞质表面的蛋白酪氨酸磷酸酶。这种酪氨酸磷酸酶在胰岛素受体（IR）活性调节中起主要作用。我们利用生物发光共振能量转移（BRET）研究了活细胞中IR与PTP1B的相互作用。将IR与海肾荧光素酶融合，将PTP1B的底物捕获突变体与绿色荧光蛋白（GFP）的黄色变体融合。当这两个伙伴相互作用时，荧光素酶和YFP之间发生能量转移，并且可以检测到YFP发出的荧光信号。IR与PTP1B的相互作用可以实时监测超过30分钟。胰岛素迅速且剂量依赖性地刺激这种相互作用。IR与PTP1B的基础（非胰岛素依赖性）相互作用在可溶性形式下比在内质网靶向形式的PTP1B下要低得多，这表明这种基础相互作用主要发生在内质网中。在基础状态下，通过共聚焦显微镜和细胞分级实验证明，PTP1B和IR确实在内质网中共定位。此外，用衣霉素抑制IR加工表明，PTP1B与IR的基础相互作用发生在内质网中IR前体的生物合成过程中。这些结果强烈表明，PTP1B不仅使已被胰岛素激活的胰岛素受体去磷酸化，而且在其生物合成过程中调节胰岛素受体前体。PTP1B定位于内质网对于防止未成熟胰岛素受体前体的非胰岛素依赖性自主活性可能很重要。
