Wang X Y, Bergdahl K, Heijbel A, Liljebris C, Bleasdale J E
Cell & Molecular Biology, Research & Development, Pharmacia & Upjohn, Kalamazoo, MI 49007, USA.
Mol Cell Endocrinol. 2001 Feb 28;173(1-2):109-20. doi: 10.1016/s0303-7207(00)00402-0.
One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.
治疗2型糖尿病核心的胰岛素抵抗的一种策略可能是将胰岛素受体（IR）维持在活性（酪氨酸磷酸化）形式。由于蛋白酪氨酸磷酸酶1B（PTP1B）会结合并随后使IR去磷酸化，PTP1B-IR结合的抑制剂是潜在的胰岛素“增敏剂”。开发了一种闪烁邻近分析（SPA）来表征和定量PTP1B-IR结合。将人IR溶解并捕获在包被有麦胚凝集素（WGA）的SPA珠上。随后，人催化无活性的[35S]PTP1B Cys(215)/Ser（PTP1B(C215S)）与凝集素锚定的IR结合，导致SPA珠产生可定量的闪烁。PTP1B与IR的结合对pH和二价阳离子敏感。当pH从6.2升高到7.8时，Ca(2+)和Mn(2+)而非Mg(2+)能显著减弱观察到的PTP1B-IR结合的丧失。PTP1B与胰岛素刺激细胞的IR的结合远大于与未刺激细胞的IR的结合，并且被抗磷酸酪氨酸抗体或用碱性磷酸酶处理IR所抑制，这表明IR的酪氨酸磷酸化是PTP1B结合所必需的。以各种IR磷酸酪氨酸结构域为模型的磷酸肽各自仅部分抑制PTP1B-IR结合，表明IR的多个结构域可能参与结合PTP1B。然而，PTP1B(C215S)对[35S]PTP1B(C215S)的竞争性置换最符合一个结合位点，其解离常数（K(d)）在100 - 1000 nM范围内，具体取决于pH和二价阳离子。PNU-200898是一种强效且选择性的PTP1B抑制剂，其在PTP1B活性位点的取向已被解析，它以相同的效力竞争性抑制催化作用和PTP1B-IR结合。这种用于PTP1B-IR结合的新型分析结果表明，PTP1B通过其活性位点优先结合酪氨酸磷酸化的IR，并且这种结合可能易受小分子的治疗性干扰。
