SITE-DIRECTED DERIVATIZATION OF INSULIN RECEPTOR

了解胰岛素受体功能的关键在于 胰岛素与受体相互作用， 信号转导 胰岛素受体是一种蛋白酪氨酸激酶， 蛋白激酶，甚至是Ser/Thr激酶， 同源性 如果有可能确定受体使用 同源基团来执行与这些激酶相同的功能， 一旦这些酶的3D结构已知，胰岛素受体 可以使用建立的地标将构象投射到其上 通过衍生化。 拟议研究的目标是建立这样的地标， 胰岛素受体选择性衍生功能重要 基团并确定衍生化位点。 的第一个问题 需要回答的是1）烷基化抑制的SH基团在哪里 受体激活？2)连接受体的S-S桥在哪里 子单位在一起？3)胰岛素在哪里与受体结合？答案 这些问题将寻求衍生一类新的 一般类型X-R-亚氨基生物素的放射性标记试剂，其中X是 衍生试剂，R是间隔基团，亚氨基生物素是"手柄"， 由此分离衍生化位点。 这些新试剂 将显著简化衍生肽的纯化。 一旦确定了衍生化氨基酸的位置， 将被前受体基因的点突变所取代。 使用这两种方法来验证特定氨基酸的重要性 对受体功能和构象的研究将提供强有力的证据， 功能上重要的地点已经确定。

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.