Regulation and Function of Urocortins and their Receptors

项目摘要/摘要 促肾上腺皮质激素释放因子(CRF)家族神经肽(CRF和urocortins[UCN]1-3)及其 受体(CRFR1、CRFR2)是中枢神经系统应激的重要介质。因此，一个 全身性抑制它们的功能并不是一个有吸引力的治疗模式。这种多肽/受体的成分 该家族在肠道中显著表达，其局部功能尚待确定。我们的 来自CRFR2杂合子小鼠的初步数据表明，CRFR2系统的扰动使 老鼠更容易受到突如其来的压力和免疫挑战。我们的结果表明，新的治疗方法 肠炎性疾病可能需要CRF受体的拮抗剂或激动剂，这取决于 受影响的肠道区域和炎症性疾病的类型。我们在结肠炎期间观察到的一个显著特征 UCN配体及其受体的mRNA和蛋白水平是相互变化的。这些差异 改变可以改变UCN诱导的受体从质膜向质膜的运输。然而， 这种向循环或降解途径的不同运输的分子机制仍未被探索。 因此，我们的假设是，UCN亚型通过CRF受体发出不同的信号，控制着 肠道炎症在分子、细胞和器官水平。这项建议的目的是： 具体目的1.明确UCN/CRFR系统在肠道炎症中的系统作用。我们会 用药理学和遗传学方法确定UCN/CRFR系统在肠道中的系统作用 发炎。我们将确定CRFR2基因敲除和杂合子小鼠是否表现出对 炎症和激动剂治疗与它们的野生型窝产仔相比。我们将确定是否需要更改 通过测量体重增加、死亡率、组织损伤和液体渗漏的变化来评估结肠炎的严重程度。 特定目的2.确定结肠特异的UCN和CRFR在肠道中的作用 使用RNA干扰(RNAi)的炎症。为了确定UCN/CRFR系统的冒号特异性作用， 我们将建立利用RNAi在结肠局部下调UCNs和CRFR2的表达。我们会 确定RNAi在结肠中的空间和时间效应，并评估结肠特异的敲除 使用特定目标1中定义的终点，UCN和CRFR的使用足以改善或加重结肠炎。 具体目的3.明确Ucn1诱导CRFR1转运的机制和功能。我们会 共聚焦显微镜观察Ucn1刺激对CRFR1亚细胞定位的影响 和免疫荧光。我们将探索接头/支架蛋白(阻滞素和支架蛋白)的能力 Dynamin)以使CRFR脱敏和复敏。我们将通过以下方式评估Ucn1对CRFR1活性的调节 检测Ucn1刺激的细胞内钙动员。因此，对尿皮质素及其受体的操纵 受体为了解其胃肠道功能提供了一个有效的起点，这可能有助于 炎症性肠病新疗法的开发。

PROJECT SUMMARY/ABSTRACT The corticotropin-releasing factor (CRF) family of neuropeptides (CRF and urocortins [Ucn] 1-3) and their receptors (CRFR1, CRFR2) are essential mediators of stress in the central nervous system. Therefore a systemic inhibition of their function is not an attractive therapeutic model. Components of this peptide/receptor family are prominently expressed within the intestine, where their local functions remain to be defined. Our preliminary data from CRFR2 heterozygous mice suggests that perturbation of the CRFR2 system renders the mice more susceptible to sudden stress and immune challenges. Our results suggest that new treatments for intestinal inflammatory diseases may require either antagonists or agonists of CRF receptors, depending upon the affected intestinal region and the type of inflammatory disease. A striking feature we observed during colitis was reciprocal changes in mRNA and protein levels of Ucn ligands and their receptors. These differential changes can alter Ucn-induced trafficking of receptors from and to the plasma membrane. However, the molecular mechanisms of this divergent trafficking to recycling or degradatory pathways remain unexplored. Thus, our hypothesis is that Ucn isoforms, differentially signaling through CRF receptors, govern intestinal inflammation at the molecular, cellular and organ levels. The aims of this proposal are to: Specific Aim 1. To define the systemic role of the Ucn/CRFR system in intestinal inflammation. We will use pharmacological and genetic approaches to define the systemic role of the Ucn/CRFR system in intestinal inflammation. We will determine if CRFR2 knockout and heterozygous mice exhibit differential sensitivity to inflammation and agonist treatment as compared with their wild type littermates. We will determine alterations in colitis severity by measuring changes in body weight gain, mortality, histological damage, and fluid leak. Specific Aim 2. To define the contributions of colon-specific Ucns and CRFRs in intestinal inflammation using RNA interference (RNAi). To determine the colon-specific role of the Ucn/CRFR system, we will establish the use RNAi to knockdown expression of Ucns and CRFR2 locally in the colon. We will determine the spatial and temporal effects of RNAi in the colon, and assess whether colon-specific knockdown of Ucns and CRFRs is sufficient to ameliorate or exacerbate colitis, using end points defined in Specific Aim 1. Specific Aim 3. To define the mechanism and function of Ucn1-induced trafficking of CRFR1. We will examine the influence of Ucn1 stimulation on the subcellular localization of CRFR1 using confocal microscopy and immunofluorescence. We will explore the ability of the adaptor/scaffolding proteins (¿-arrestins and dynamin) to desensitize and resensitize CRFRs. We will assess Ucn1 regulation of CRFR1 activity by measuring Ucn1-stimulated intracellular Ca2+ mobilization. Thus, manipulation of the urocortins and their receptors provides an effective starting point for understanding their GI functions, which could contribute to the development of new treatments for inflammatory bowel disease.