Construction of Molecular Map of Pancreatic beta-cell.

了解胰腺β细胞分泌胰岛素的机制将为了解糖尿病和低血糖的发病机制提供线索。我们重点研究了在胰腺β细胞中表达的编码蛋白的基因，这些蛋白在葡萄糖诱导的胰岛素分泌中参与刺激-分泌偶联。我们发现了两个新的基因Kir6.2和Noc2，它们在胰岛素分泌中起重要作用。我们已经研究了这些分子的结构和功能。结果总结如下：1。atp敏感的K^+通道。我们克隆了一个内向整流器K^+通道族的新成员Kir6.2。Kir6.2是一种由390个氨基酸组成的蛋白质，有两个跨膜片段。通过对Kir6.2和sulfonylureas受体（SUR1）的共表达研究，我们发现胰腺β细胞atp敏感的K^+通道包括Kir6.2和SUR1。SUR1广泛用于治疗非胰岛素依赖型糖尿病。SUR1对药物和细胞核的敏感性更高，而Kir6.2形成K^+离子可渗透的孔域。SUR1的同工异构体SUR2也被克隆出来。我们已经证明，SUR1基因或Kir6.2基因突变可引起婴儿期家族性低血糖。我们已经在胰腺β细胞中产生了表达K_<ATP>通道亚基Kir6.2的显性阴性形式的转基因小鼠[Kir6.2 g132s，在132位用丝氨酸（Ser）取代甘氨酸（Gly）]。Kir6.2G132S转基因小鼠出现新生儿低血糖伴高胰岛素血症，成年高血糖伴低胰岛素血症及β细胞群减少。转基因小鼠在出现高血糖之前，β细胞凋亡的频率较高，这表明K_<ATP>通道除了调节胰岛素分泌外，还可能在β细胞存活中起重要作用。Noc2。我们克隆了一个含有302个氨基酸的新蛋白cDNA（命名为Noc2， no2结构域），该蛋白与Rab3A的靶分子rabphilin-3A的n端区具有40.7%的氨基酸同源性和77.9%的相似性。然而，与rabphilin-3A不同，Noc2缺乏被认为与Ca^<2+>和磷脂相互作用的两个C2结构域。Noc2主要表达于内分泌组织，包括胰腺β细胞。胰岛素分泌细胞系MIN6亚细胞部分的免疫印迹分析和免疫细胞化学显示，Noc2是一个38 kDa的蛋白，存在于细胞质中。在共转染生长激素（GH）的PCl2细胞中，Noc2的过表达增强了高K^+诱导的GH分泌。酵母双杂交系统筛选表明，Noc2与含有LIM结构域的蛋白zyxin（细胞骨架的一个组成部分）相互作用，并通过共免疫沉淀实验进一步证实了这种相互作用。因此，Noc2可能通过与细胞骨架相互作用参与胰腺β细胞的胞吐调节。少

Understanding of the mechanisms of insulin secretion from pancreatic beta-cells should provide a clue to understand the pathogenesis of diabetes mellitus and hypoglycemia.We have focused the genes encoding proteins expressed in pancreatic beta-cells that are involved in stimulus-secretion coupling in glucose-induced insulin secretion. We have identified two novel genes, Kir6.2 and Noc2, which play important roles in insulin secretion. We have studied the stuctures and functions of these molecules.The results are summarized as follows.1.ATP-sensitive K^+ channels. We have cloned a novel member of the inward rectifier K^+ channel family, Kir6.2. Kir6.2 is a protein of 390 amino acids having two transmembrane segments. By coexpression studies of Kir6.2 and a receptor for sulfonylureas (SUR1), widely used in the treatment of non-insulin dependent diabetes mellitus, we have shown that pancreatic beta-cell ATP-sensitive K^+ channel comprises Kir6.2 and SUR1. While SUR1 confers drug and nucle … More otide sensitivities, Kir6.2 forms K^+ ion permeable pore domain. An isoform of SUR1, SUR2, was also cloned. We have shown that mutations of SUR1 gene or Kir6.2 gene cause familial hypoglycemia of infancy. We have generated transgenic mice expressing a dominant-negative form of the K_<ATP> channel subunit Kir6.2 [Kir6.2G132S,substitution of glycine (Gly) with serine (Ser) at position 132] in pancreatic beta-cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta-cell population in adults. The transgenic mice exhibited a high frequency of apoptotic beta-cells prior to the appearance of hyperglycemia suggesting that the K_<ATP> channel might play a significant role in beta-cell survival in addition to its role in the regulation of insulin secretion.2. Noc2. We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, noC2domain)which has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A,a target molecule of Rab3A.However, unlike rabphilin-3A,Noc2 lacks two C2 domains that are thought to interact with Ca^<2+> and phospholipids. Noc2 is expressed predominantly in endocrine tissues including pancreatic beta-cells. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38 kDa protein present in the cytoplasm. Overexpression of Noc2 in PCl2 cells cotransfected with growth hormone (GH) enhances high K^+-induced GH secretion. Screeniy with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in pancreatic beta-cells by interacting with the cytoskeleton. Less