Crystallization and structural analysis of the human insulin receptor

人胰岛素受体(HIR)是由两个α亚基(735个氨基酸，135 kDa)和两个β亚基(620个氨基酸，95 kDa)组成的糖蛋白。HIR形成二硫键连接的异四聚体(β-α-α-β)。该受体的胞外区由整个α亚基和预测跨膜区外的β亚基的一部分组成。利用截短型人胰岛素受体(HIR)的表达载体，我们构建了一个稳定的中国仓鼠卵巢(CHO)细胞系，该细胞系能分泌His标记的α亚单位(胰岛素结合区)。为了检测His标记的HIRα的特性，我们纯化了CHO细胞分泌的蛋白质。His标记的hirα被糖基化并加工成二聚体。该分子与胰岛素结合的亲和力与完整的HIR相似。His标记的全长HIR在胰岛素刺激下在CHO细胞中被自动磷酸化，我们将纯化大量的IRα，并将其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.