MODULATION OF INSULIN RECEPTOR FUNCTION

工作总结：在完整细胞中，添加一个与胰岛素受体羧基末端的离散区域相对应的甾烷基偶联合成肽可增强胰岛素介导的反应。然而，硬脂酰偶联肽和膜脂双分子层之间的疏水相互作用引入了一个二维反应环境，从而使效率和特异性可能比自由扩散肽低（或高）个数量级。因此，我们尝试使用minigene方法稳定表达胰岛素受体羧基端相同片段，并评估其对完整细胞中胰岛素和IGF-1反应的影响。我们的研究表明，minigene的稳定表达可以增加胰岛素诱导的胰岛素受体自磷酸化，与空载体转染的细胞相比，其自磷酸化水平提高了2.4倍。这伴随着一些信号中间体激活的显著增加。重要的是，我们发现minigene的表达对胰岛素的作用具有高度选择性，因为它对胰岛素样生长因子I受体激酶的活性和功能没有影响。minigene激活胰岛素受体催化功能的分子决定因素尚不清楚。然而，我们认为minigene[编码受体羧基端序列特异性的15个氨基酸片段]与受体β亚基的非催化区域结合，从而引起有利于催化的构象变化。评估这一假设的工作正在进行中。

Summary of work: The addition of a stearyl-conjugated synthetic peptide that corresponds to a discrete region within the insulin receptor carboxyl terminus results in the potentiation of insulin-mediated responses in intact cells. However, the hydrophobic interaction between stearyl conjugated peptides and the membrane lipid bilayer introduces a two-dimensional reaction environment, thus enabling efficiency and specificity that may be orders of magnitude lower (or higher) than would be possible with freely diffusing peptide. Therefore, we have attempted to express stably the same fragment of the insulin receptor carboxyl terminus using a minigene approach and to assess its effect toward insulin and IGF-1 responses in intact cells. Our study showed that stable expression of the minigene can increase insulin-induced insulin receptor autophosphorylation that was 2.4-fold higher when compared to that of cells transfected with empty vector. This was accompanied by a significant increase in activation of a number of signaling intermediates. Importantly, it was found that expression of the minigene was highly selective to insulin's action as it had no effect toward insulin-like growth factor I receptor kinase activity and function. The molecular determinant for activation of the insulin receptor catalytic function by the minigene remains unclear. However, we believe that the minigene [which encodes a sequence-specific 15 amino acid fragment of the receptor carboxyl terminus] binds with a non-catalytic region of the receptor beta-subunit and thus causes a conformational change that facilitates catalysis. Work is underway to assess this hypothesis.