Modulation of PKC Response and Insulin Secretion by the Beta Subunit of Calcium C

描述(申请人提供)：根据血糖浓度，胰岛素在维持血糖稳态中起主要作用。然而，胰岛素的释放与血糖浓度无关，会导致低血糖，这通常发生在使用现有的抗糖尿病药物时。因此，理想的治疗方法 对于糖尿病来说，就是要根据血糖水平增加胰岛素浓度。识别这种现象是我们实验室的长期目标。电压门控钙通道(CAV)�3亚单位基因敲除(�3-/-)小鼠的葡萄糖依赖性胰岛素分泌增加。CAv�基因敲除诱导的胰岛素分泌增加被称为负调节，因为Cav�亚单位可能在可用时抑制胰岛素分泌。CAV�亚基是CAV通道的一个辅助亚基，属于膜激活鸟苷酸激酶(MAGUK)家族，在通道周围的多条信号通路中起着独立的支架作用。因此，CAV�亚基可能通过与参与胰岛素分泌的中介相互作用而负向调节胰岛素的分泌。识别CAV�亚基的靶标并了解其负性调节胰岛素分泌的机制是本研究的目的。CAV�亚基在功能上是相连的 Cav通道和蛋白激酶C(PKC)家族的同工酶是胰岛素分泌的两种促分泌剂。这些亚基对于Cav�1亚基的运输和它们在膜上作为Cav通道的组装以及它们被PKC的调节是必需的。因此，CAV�1亚基和PKC同工酶或两者都可能是CAV�亚基负性调节胰岛素分泌的靶标。我们将通过抑制Cav�亚基、检测胰岛素释放、Cav通道活性和PKC同工酶来研究负调控。在这方面，我们将使用小干扰RNA(SiRNA)来沉默选定的Cav亚基。具体目的I将确定调节胰岛素分泌的CAV�亚基(�1-�4)以及与其相关的CAV�激活的PKC同工酶(cPKCs、npkcs和apkcs)。具体目标II将确定与所涉及的CAV�和PKC同工酶相互作用的CAV通道(CAV1.2、1.3、2.1和2.3)。这两个特定目标将使用在小鼠体内的研究和使用小鼠培养的胰腺�细胞的体外研究。第三部分将利用非洲爪哇卵母细胞表达系统研究�-1亚基和�亚基之间的相互作用机制及同工酶。具体地说，这一目标将确定CAV�1亚基的�和PKC同工酶靶点的丝氨酸/苏氨酸蛋白C磷酸化位点，并研究这三种蛋白之间的相互作用机制。

DESCRIPTION (provided by applicant): Insulin plays the major role in the maintenance of glucose homeostasis based on the concentration of blood glucose. However, the release of insulin independent of blood glucose concentration will lead to hypoglycemia which often takes place with the administration of currently available antidiabetic drugs. Hence, the ideal treatment for diabetes is to increase insulin concentration based on blood glucose level. The identification of such a phenomenon is the long-term goal of our laboratory. Glucose-dependent insulin secretion is increased in voltage-gated Ca2+-channel (Cav) �3 subunit knockout (�3-/-) mice. The Cav�-knockout-induced increase of insulin secretion is called negative modulation since Cav� subunits may be inhibiting insulin secretion when they are available. Cav� subunits, in addition to being an auxiliary subunit of Cav channels, act independently in scaffolding multiple signaling pathways around the channel since they belong to the membrane activated guanylate kinase (MAGUK) family. Hence it is possible that Cav� subunits negatively modulated insulin secretion by interacting with the intermediaries involved in insulin secretion. The identification f the targets of Cav� subunits and the understanding of the mechanisms leading to their negative modulation of insulin secretion is the aim of this proposal. Cav� subunits are functionally linked to Cav channels and protein kinase C (PKC) family of isozymes, the two secretagogues of insulin secretion. These subunits are required for the trafficking of Cav �1 subunits and their assembly as Cav channels in the membrane and their modulation by PKC. Hence, it is likely that either Cav �1 subunits and PKC isozymes or both are the targets of Cav� subunits in their negative modulation of insulin secretion. We will study the negative modulation by inhibiting the Cav� subunits and examining the insulin release, the activity of Cav channels and PKC isozymes. In this regard, we will use small interfering RNA (siRNA) to silence the selected Cav subunits. The Specific Aim I will identify the Cav� subunits (�1- �4) that modulated insulin secretion and the PKC isoymes (cPKCs, nPKCs and aPKCs) that the implicated Cav� activated. The Specific Aim II will identify the Cav channels (Cav 1.2, 1.3, 2.1 and 2.3) with which the implicated Cav� and PKC isozymes interact. These two Specific Aims will use in vivo studies in mice and in vitro studies employing mouse cultured pancreatic -� cells. The Specific Aim III will study the mechanism of interaction between the implicated Cav �1 and � subunits and the PKC isozymes employing Xenopus oocytes expression system. To be specific, this Aim will identify the Ser/Thr PKC phosphorylation sites of Cav �1 subunits that are targeted by the Cav� and PKC isozymes and study the mechanism of interaction between these three proteins.