SITE-DIRECTED DERIVATIZATION OF INSULIN RECEPTOR

了解胰岛素受体功能的关键在于 现在认识到胰岛素与受体相互作用导致 信号转导。胰岛素受体是一种蛋白酪氨酸激酶， 蛋白激酶，甚至丝氨酸/苏氨酸激酶，都有相同的序列 同源。如果有可能确定受体使用 同源基团执行与这些激酶相同的功能，然后 一旦知道了这些酶的3D结构，胰岛素受体 可以使用所建立的地标将构象投射到其上 通过衍生化。 拟议研究的目标是在 具有重要功能的胰岛素受体选择性衍生化 群和确定衍生化的位置。第一个问题 有待回答的是1)烷基化抑制的SH基团在哪里 受体激活？2)与受体结合的S-S桥在哪里 亚基在一起？3)胰岛素在哪里与其受体结合？答案 将通过派生出一类新的 普通型X-R-亚胺生物素的放射性标记试剂，其中X是 衍生化试剂，R为间隔基，亚胺生物素为“把手” 用它来分离衍生的位置。这些新试剂 将极大地简化衍生肽的纯化。 一旦确定了衍生氨基酸的位置，它们 Wi.U被前受体的基因点突变所取代。 使用这两种方法来验证特定氨基酸的重要性 受体的功能和构象将提供强有力的证据 已经确定了具有重要功能的地点。

The key to understanding the function of the insulin receptor lies in recognizing now insulin interacts with the receptor to bring about signal transduction. Insulin receptor is a protein tyrosine kinase and protein kinases, even Ser/Thr kinases, share certain sequence homologies. If it is possible to establish that the receptor uses homologous groups to perform the same functions as these kinases, then once a 3D structure is known for these enzymes, the insulin receptor conformation can be projected onto it using the landmarks established through derivatization. The goal of the proposed research is to establish such landmarks on the insulin receptor by selective derivatization of functionally important groups and determining the site of derivatization. The first questions to be answered are 1) Where is the SH group whose alkylation inhibits receptor activation? 2) Where are the S-S bridges that bind receptor subunits together? 3) Where does insulin bind to its receptor? Answers to these questions will be sought by derivatizing with a new class of radiolabeled reagents of the general type X-R-iminobiotin where X is a derivatizing reagent, R is a spacer group, and iminobiotin is a "handle" by which the derivatized sites wi.U be isolated. These new reagents will dramatically simplify the purification of derivatized peptides. Once the location of the derivatized amino acids is established, they wi.U be replaced by point mutations of the gene for the proreceptor. Using both approaches to validate the importance of specific amino acids for receptor function and conformation will provide strong evidence that functionally important sites have been identified.