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理解糖尿病和低血糖的发病机制提供线索。我们重点研究了在葡萄糖诱导的胰岛素分泌过程中，参与刺激-分泌偶联的胰岛β细胞表达的蛋白质编码基因。我们已经鉴定出两个新的基因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型K^+通道家族的一个新成员Kir6.2。Kir6.2是一个由390个氨基酸组成的蛋白质，有两个跨膜片段。通过对Kir6.2和磺脲类受体(SUR1)的共表达研究，我们发现胰腺β细胞ATP敏感性K~+通道由Kir6.2和SUR1组成。而SUR1授予药物和核…更多的核苷酸敏感性，Kir6.2形成K~(++)离子可渗透的孔区。同时克隆了SUR1的一个亚型SUR2。我们已经证明，SUR1基因或Kir6.2基因的突变会导致婴儿时期的家族性低血糖。我们已经产生了在胰腺β细胞中表达显性-阴性形式的K_&lt；ATP&gt；通道亚单位Kir6.2[Kir6.2G132S，132位用丝氨酸(Ser)取代甘氨酸(Gly)]的转基因小鼠。Kir6.2G132S转基因小鼠在新生儿出现低血糖伴高胰岛素血症，在成人出现高血糖伴低胰岛素血症和β细胞数量减少。在高血糖出现之前，转基因小鼠表现出高频率的β细胞凋亡，这表明K&lt；ATP&gt；通道除了在调节胰岛素分泌方面的作用外，还可能在β细胞的存活中发挥重要作用。2号。我们克隆了一个编码302个氨基酸的新蛋白(命名为NOC2，noC2结构域)，它与Rab3A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。但是，与RabPhilin-3A不同的是，NOC2缺少两个C2结构域，这两个C2结构域被认为与钙离子和磷脂相互作用。NOC2主要在包括胰岛β细胞在内的内分泌组织中表达。免疫印迹分析和免疫细胞化学分析表明，NOC2是存在于细胞质中的一种38 kDa的蛋白质。Noc2基因在共转染生长激素(GH)的PCl2细胞中的过表达增强了高K~(++)诱导的GH分泌。与酵母双杂交系统的筛选表明，NOC2与细胞骨架中含有LIM结构域的蛋白Zysin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，Noc2可能通过与细胞骨架的相互作用参与调节胰岛β细胞的胞吐。较少

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