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

描述（由申请方提供）：胰岛素在基于血糖浓度的葡萄糖稳态维持中起主要作用。然而，胰岛素的释放不依赖于血糖浓度，这将导致低血糖症，这通常在施用目前可用的抗糖尿病药物时发生。因此，理想的治疗方法 治疗糖尿病的方法是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。 葡萄糖依赖性胰岛素分泌在电压门控Ca 2+通道（Cav）<$3亚基敲除（<$3-/-）小鼠中增加。The Cavé- 敲除诱导的胰岛素分泌增加被称为负调节，因为Cav亚基在可用时可能抑制胰岛素分泌。卡夫亚基除了作为Cav通道的辅助亚基之外，还独立地在通道周围的多个信号传导途径中起支架作用，因为它们属于膜活化鸟苷酸激酶（MAGUK）家族。因此，Cav亚基可能通过与参与胰岛素分泌的中间体相互作用来负调节胰岛素分泌。本研究的目的是确定Cav亚基的靶点并了解其对胰岛素分泌负调节的机制。 Cav亚基在功能上与 Cav通道和蛋白激酶C（PKC）家族的同工酶，这两种促胰岛素分泌素。这些亚基是Cav 1亚基运输和它们在膜中作为Cav通道组装以及它们通过PKC调节所必需的。因此，很可能Cav <$1亚基和PKC同工酶或两者都是Cav <$1亚基负调节胰岛素分泌的靶点。我们将通过抑制Cav亚基和检测胰岛素释放、Cav通道活性和PKC同工酶来研究负性调节。在这方面，我们将使用小干扰RNA（siRNA）来沉默所选择的Cav亚基。 特异性目的I将鉴定调节胰岛素分泌的Cav <$亚基（<$1-<$4）和相关Cav <$激活的PKC同工酶（cPKC、nPKC和aPKC）。Specific Aim II将识别Cav通道（Cav 1.2、1.3、2.1和2.3），其中涉及Cav和PKC同工酶相互作用。这两个特定目标将使用小鼠体内研究和使用小鼠培养的胰腺细胞的体外研究。Specific Aim III将使用爪蟾卵母细胞表达系统研究相关Cav â 1和â亚基与PKC同工酶之间相互作用的机制。具体而言，本研究将鉴定Cav <$1亚基的Ser/Thr PKC磷酸化位点，并研究这三种蛋白质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.