The role of phosphorylation in regulating the antidiabetic effects of O3FAR-1

描述(由申请人提供)：omega-3脂肪酸受体(O3FAR1)，通常也被称为GPR120，是一种G蛋白偶联受体(GPCR)，已被证明可被长链不饱和脂肪酸，特别是那些属于omega-3脂肪酸(O3FA)家族的脂肪酸所激活。由于这种受体能够刺激肠道分泌胰升糖素样肽-1(GLP-1)，从而刺激胰腺b细胞分泌胰岛素，从而降低血糖，因此引起了人们的极大兴趣。除了这种作用外，最近还证明，O3FAR1的激动剂通过抑制NF-KB和JNK致炎通路而产生深刻的抗炎作用，而这些通路在巨噬细胞中被激活时，会导致胰岛素抵抗、体重增加和2型糖尿病。我们的实验室发现，人类存在两种O3FAR1亚型，O3FAR1-Long和Short，这是mRNA选择性剪接的结果，我们的结果表明，这两种亚型受到受体磷酸化的不同调控。我们的初步数据还表明，G蛋白偶联受体激酶-6(GRK6)负责O3FAR1亚型的同源(激动剂诱导)磷酸化。重要的是，O3FAR1的抗糖尿病作用依赖于b-arrestin-2伙伴蛋白，而b-arrestin-2蛋白仅被GRK磷酸化的受体所招募，这表明受体磷酸化驱动O3FAR1的抗糖尿病活性。此外，我们的结果表明，调节受体表达和激动剂敏感性的O3FAR1的基础磷酸化是由蛋白激酶C(PKC)介导的，并且PKC对两种O3FAR1亚型的影响是不同的。我们提出了两个特定的目标，这将为O3FAR1的激活和信号调节机制提供结构性指导，也将有助于更好地理解两种O3FAR1亚型之间的信号差异。在特定的目标一，我们将使用蛋白质组驱动的方法定位GRK和PKC介导的O3FAR1亚型磷酸化的特定位点。在特定的目标二中，我们将评估这些磷酸化机制在调节受体与b-arrestin-2的相互作用，激活Gaq/11-磷脂酶C/Ca+2信号通路，分泌GLP-1，以及通过抑制JNK和NF-KB途径产生抗炎作用方面的功能意义。总之，这些结果将为所有已知的O3FAR1功能的磷酸调节机制提供结构性的见解。由于美世大学药学与健康科学学院的研究活动完全依赖于学生的融合，该项目将允许将研究生(博士)、专业(药学)和本科生纳入一个既定的重要研究项目，并将极大地加强研究环境，与地区资助机制的目标保持一致。

DESCRIPTION (provided by applicant): The omega-3 fatty acid receptor (O3FAR1), also commonly referred to as GPR120, is a G protein-coupled receptor (GPCR) that has been shown to be agonized by long chained unsaturated fatty acids, specifically, those belonging to the omega-3 fatty acid (O3FA) family. This receptor has generated considerable interest due to its ability to stimulate intestinal secretion of the incretin hormone glucagon-like-peptide-1 (GLP-1), which stimulates pancreatic b-cells leading to insulin secretion and subsequently, decreases in blood glucose. In addition to this effect, it has recently been demonstrated that agonism of O3FAR1 leads to profound anti- inflammatory effects via inhibition of the NF-KB and JNK proinflammatory pathways, which when otherwise activated in macrophages, lead to insulin resistance, weight gain, and type 2 diabetes. Our laboratory has revealed that two human O3FAR1 isoforms, O3FAR1-long and short, exist as a consequence of alternative mRNA splicing and our results suggest that these isoforms are differentially regulated by receptor phosphorylation. Our preliminary data also show that G protein-coupled receptor kinase-6 (GRK6) is responsible for homologous (agonist-induced) phosphorylation of O3FAR1 isoforms. Importantly, the antidiabetic effects of O3FAR1 have been shown to be dependent on b-arrestin-2 partner proteins, which are recruited only to GRK-phosphorylated receptors, suggesting that receptor phosphorylation drives O3FAR1 antidiabetic activity. Additionally, our results demonstrate that basal phosphorylation of O3FAR1, which regulates receptor expression and agonist sensitivity, is mediated by protein kinase C (PKC), and that there are differential affects of PKC on the two O3FAR1 isoforms. We propose two specific aims that will provide structural guidance on mechanisms of O3FAR1 activation and signal regulation, and will also provide a better understanding of signaling differences between the two O3FAR1 isoforms. In specific aim one, we will localize the specific sites of GRK and PKC mediated phosphorylation of O3FAR1 isoforms using a proteomic- driven approach. In specific aim two, we will assess the functional significance of these phosphorylation mechanisms with respect to their ability to regulate receptor interactions with b-arrestin-2, activate the Gaq/11- phospholipase C/Ca+2 signaling pathway, secrete GLP-1, and produce anti-inflammatory effects via inhibition of JNK and NF-KB pathways. Collectively, these results will provide structural insight into mechanisms of phospho-regulation of all known O3FAR1 functions. Since research activities at Mercer University's College of Pharmacy and Health Sciences are fully dependent on student integration, this project will allow for incorporation of graduate (PhD), professional (PharmD), and undergraduate students within an established and significant research project, and will greatly strengthen the research environment, consistent with the goals of the AREA funding mechanism.