The gastrintestinal physiology of cholecystokinin

胆囊收缩素的胃肠生理学

• 批准号: 7047786
• 负责人: JOSEPH R REEVE
• 金额: $ 27.07万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7047786
• 关键词: acinar cell; bioassay; cell surface receptors; cholecystokinin; conformation; gastrointestinal system; hormone biosynthesis; hormone receptor; laboratory rat; ligands; physiology; protein structure function; receptor binding

DESCRIPTION (provided by applicant): Producing ligands to G protein coupled receptors (GPCRs) with the desired actions is one of the most active areas of research for therapeutic agents. Determining how GPCRs, like the CCK-A receptor, interact at the molecular level with their ligands will aid in the design of reagents for treating obesity, gallbladder disease and motility disorders. CCK-58 is a major endocrine form of cholecystokinin that has different biological activity from CCK-8. One major difference is that CCK- 58 does not induce pancreatitis at any dose while high CCK-8 is an accepted model for induction of pancreatitis. There is evidence that peptide ligands interact with their GCPR via a lipid-associated mechanism. Therefore, we hypothesize that different lipid-induced tertiary structures of cholecystokinin agonists determine the biological responses elicited via the CCKA receptor. A corollary is that the N-terminus of CCK-58 acts as a scaffold for the C-terminal active region when associated with lipids. Initially we will identify intramolecular associations of the N- and C-terminus of CCK-58 and test if these associations are important for function (CCK-A receptor binding and stimulation of pancreatic protein from acinar cells). Next the lipid-associated tertiary structures of six cholecystokinin analogs (CCK-8, CCK-33, JMV-180, CCK-58, CCK-8(ns) and CCK-58(ns)) will be determined. The six analogs will be tested for differences in their physiological actions and their capacity to cause pancreatitis in an awake rat model. We will identify the 3D conformation(s) that elicit a normal physiological response or pancreatitis. Next we will titrate the receptor segment (extracellular loop 3, amino acids 345-373) with the cholecystokinin agonists and determine the specific ligand-receptor interactions. Further we will determine 3D conformation of CCK-8 and CCK-58 bound to the receptor fragment. The data collected will be used to predict amino acid substitutions that could increase binding of agonists to the CCK-A receptor. We will mutate the receptor with substitutions predicted to enhance binding to test our model.

描述（由申请人提供）：与G蛋白偶联受体（GPCR）产生配体具有所需的作用是治疗剂最活跃的研究领域之一。确定GPCR（例如CCK-A受体）如何在分子水平上与配体相互作用将有助于设计用于治疗肥胖，胆囊疾病和运动障碍的试剂。 CCK-58是一种主要内分泌形式的胆囊化蛋白，其生物学活性与CCK-8不同。一个主要区别是CCK-58不会在任何剂量下诱导胰腺炎，而高CCK-8是诱导胰腺炎的公认模型。有证据表明，肽配体通过脂质相关机制与GCPR相互作用。因此，我们假设胆囊动蛋白激动剂的不同脂质诱导的三级结构决定了通过CCKA受体引起的生物学反应。推论的是，与脂质相关时，CCK-58的N末端是C端活动区域的支架。最初，我们将确定CCK-58 N和C末端的分子内关联，并测试这些关联对功能是否重要（CCK-A受体结合和刺激来自腺泡细胞的胰腺蛋白）。接下来，将确定六种胆囊动蛋白类似物（CCK-8，CCK-33，JMV-180，CCK-58，CCK-58，CCK-8（NS）和CCK-58（NS））的脂质相关第三纪结构。这六个类似物将测试其生理作用的差异以及在清醒大鼠模型中引起胰腺炎的能力。我们将确定引起正常生理反应或胰腺炎的3D构型。接下来，我们将用胆囊基蛋白激动剂滴定受体段（细胞外环3，氨基酸345-373），并确定特定的配体 - 受体相互作用。此外，我们将确定与受体片段结合的CCK-8和CCK-58的3D构象。收集的数据将用于预测可能增加激动剂与CCK-A受体结合的氨基酸取代。我们将用预测的替换来突变受体，以增强结合以测试我们的模型。