Crystallization and structural analysis of the human insulin receptor

人胰岛素受体（hIR）是一种糖蛋白，由两个α亚基（735个氨基酸; 135 kDa）和两个β亚基（620个氨基酸; 95 kDa）组成，通过蛋白水解加工从单一多肽衍生而来（Cell，1985，Ebina et al.）。hIR形成二硫键连接的异源四聚体（β-α-α-β）。受体的胞外区由整个α亚基和预测的跨膜结构域外部的部分β亚基组成。利用截短型人胰岛素受体（human insulin receptor，hIR）的表达载体，构建了稳定分泌hIR α亚基（insulin binding domain，hIR α亚基）的中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞系。为了检测His标记的hIRα的特性，我们纯化了从CHO细胞分泌的蛋白。His标记的hIRα被糖基化并加工成二聚体。该分子以与完整hIR相似的亲和力结合胰岛素。胰岛素刺激CHO细胞使hIR自磷酸化，纯化大量的IRα，与胰岛素共结晶，并测定其晶体结构。

The human insulin receptor (hIR) is a glycoprotein composed of two α subunits (735 amino acid ; 135kDa) and two β subunits (620 amino acid ; 95kDa) derived by proteolytic processing from a single polypeptide (Cell, 1985, Ebina et al.). The hIR forms a disulfide-linked heterotetramer (β-α-α-β). The extracellular region of the receptor consists of the entire α subunit and a portion of the β subunit external to the predicted transmembrane domain. Insulin binds to the α subunit and activates tyrosine kinase of the intracellular β subunit to transmit the insulin signal.Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged α subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRα, we purified the protein secreted from the CHO cells. The His-tagged hIRα was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells.We will purify the large amount of IRα, co-crystalize it with insulin and determine the crystal structure.