Construction of Molecular Map of Pancreatic beta-cell.

了解胰岛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。我们集中研究了在葡萄糖诱导的胰岛素分泌中参与刺激-分泌偶联的胰岛β细胞中表达的蛋白质的基因编码。我们已经确定了两个新的基因，Kir6.2和Noc 2，它们在胰岛素分泌中起重要作用。我们对这些分子的结构和功能进行了研究，主要结果如下：1. ATP敏感性K^+通道。我们克隆了内向整流钾离子通道家族的新成员Kir6.2。Kir6.2是具有两个跨膜区段的390个氨基酸的蛋白质。通过Kir6.2和磺脲类受体（SUR 1）的共表达研究，我们发现胰腺β细胞ATP敏感性K^+通道包含Kir6.2和SUR 1。磺脲类受体广泛用于治疗非胰岛素依赖型糖尿病。而SUR 1赋予药物和核 ...更多信息 K_（6.2）形成K^+渗透孔隙域。还克隆了SUR 1的一种同工型SUR 2。我们已经证明SUR 1基因或Kir6.2基因突变引起婴儿家族性低血糖症。我们<ATP>在胰腺β细胞中建立了K_通道亚基Kir6.2 [Kir6.2G132S，132位甘氨酸（Gly）被丝氨酸（Ser）取代]显性阴性表达的转基因小鼠。Kir6.2G132S转基因小鼠在新生儿中发生低血糖伴高胰岛素血症，在成人中发生高血糖伴低胰岛素血症和β细胞群减少。转基因小鼠在出现高血糖之前就表现出高频率的β细胞凋亡，提示K通道<ATP>除了在胰岛素分泌的调节中起作用外，还可能在β细胞存活中起重要作用.无2。我们克隆了一个编码302个氨基酸的新蛋白（命名为Noc 2，noC 2结构域），该蛋白与Rab 3A的靶分子rabphilin-3A的N-末端区域有40.7%的氨基酸同源性和77.9%的相似性，但与rabphilin-3A不同，Noc 2缺少两个与Ca^2+和磷脂相互作用的C2结构域。Noc 2主要在包括胰腺β细胞在内的内分泌组织中表达。胰岛素分泌细胞系MIN 6的亚细胞组分的免疫印迹分析和免疫细胞化学显示，Noc 2是存在于细胞质中的38 kDa蛋白。在与生长激素（GH）共转染的PCl 2细胞中过表达Noc 2可增强高K^+诱导的GH分泌。酵母双杂交系统筛选结果表明，Noc 2与细胞骨架中含有LIM结构域的蛋白质zyxin相互作用，免疫共沉淀实验进一步证实了这种相互作用。因此，Noc 2可能通过与细胞骨架相互作用而参与胰腺β细胞中调节的胞吐作用。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