Studies of a Novel CCK-B/Gastrin Receptor Splice Variant

新型 CCK-B/胃泌素受体剪接变体的研究

• 批准号: 6517796
• 负责人: MARK R HELLMICH
• 金额: $ 25.43万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517796
• 关键词: biomarker; calcium flux; carcinogenesis; cell growth regulation; cholecystokinin; colorectal neoplasms; gastrins; genetically modified animals; hormone receptor; human tissue; laboratory mouse; neoplastic growth; neuropeptide receptor; protein isoforms; protein structure function; protooncogene; receptor expression; receptor sensitivity; tissue /cell culture; transfection

DESCRIPTION (Applicant's Abstract): Colorectal cancer is the third leading cause of cancer death in the United States, Colon carcinogenesis is a complex, multi-step process involving progressive changes in signaling pathways regulating intestinal epithelial cell proliferation, differentiation and programmed death. The peptide hormone, gastrin 1-17 (G-17), and its non-amidated precursor, glycine-extended gastrin (G-GIy), exert potent trophic effects on colon cancer cells. The long-term goal is to understand the role of these peptide hormones in the regulation of epithelial cell biology and colon carcinogenesis. Although the growth-promoting effect of these peptides on colon cancers has been extensively documented, the identity of the receptors and intracellular signaling pathways involved remain controversial. The investigators have identified and isolated the cDNA for a novel splice variant of the human cholecystokinin-B/gastrin receptor (CCK-BR), a member of the G protein-coupled receptor (GPCR) superfamily. The splice variant (designated CCK-BRi4sv for intron 4 containing splice variant) encodes a receptor protein containing 69 additional amino acid residues in its putative third intracellular loop domain. CCK-BRi4sv is expressed in adenomatous polyps and colorectal cancers, but not in nonmalignant colonic mucosa adjacent to the cancer. Mouse Balb3T3 cells expressing the splice variant exhibited spontaneous, ligand-independent, oscillatory, increases in [Ca2+]i whereas, the same cells expressing wild-type CCK-BR (CCK-BRwt) did not. Similarly, primary cultures of human cells isolated from freshly resected colorectal cancers exhibited, ligand-independent, oscillatory increases in [Ca2+]. For both Balb3T3 and primary tumor cells, application of G-17 (10 and 200 nM, respectively) caused an increase in [Ca2+]i. Selective CCK-BR antagonists blocked the G- 17-stimulated Ca2+ responses, but not the spontaneous [Ca2+]i oscillations. In addition to spontaneous intracellular signaling, BaIb3T3 cells expressing CCK-BRi4sv exhibited an increased rate of cell proliferation (approximately 2.5-fold), in the absence of G-17, compared to cells expressing wild-type CCK-BR (CCK-BRwt). Based on these findings, the PI hypothesizes that CCK-BRi4sv may regulate colorectal cancer cell growth through both a gastrin-independent and -dependent mechanism and thus play a significant role in colorectal carcinogenesis. Furthermore, the PI hypothesizes that the function of CCK-BRi4sv in colorectal cancer biology is a direct consequence of the structural changes in the third intracellular loop domain, caused by intron retention, and the impact of those changes on intracellular signal transduction. To examine these hypotheses they plan experiments with the following specific aims: 1) to determine the spatial and temporal expression of the CCK-BR splice variant in adenomatous polyps and colon cancers; 2) to determine the effects of intron retention on receptor-mediated intracellular signal transduction and receptor desensitization/internalization; and 3) to determine the effects of ectopic expression of the CCK-BR splice variant on colonic epithelial cell homeostasis and susceptibility to carcinogen-induced colon cancer using a transgenic mouse model. These studies will provide important and new information regarding the role of the novel receptor splice variant and G-17 and G-Gly in epithelial cell biology and colon carcinogenesis. Furthermore, these studies may, in the future, provide the basis for the development of innovative therapeutic strategies for the treatment of peptide hormone-sensitive cancers.

描述（申请人的摘要）：大肠癌是第三大领先 美国癌症死亡的原因，结肠癌发生是一个复杂的 多步骤过程涉及信号通路的逐步变化 调节肠上皮细胞增殖，分化和 编程死亡。肽激素，胃蛋白1-17（G-17），及其 非体形前体，甘氨酸延伸的胃蛋白（G-GIY），发挥有效的营养 对结肠癌细胞的影响。长期目标是了解 这些肽激素在调节上皮细胞生物学和结肠中 致癌作用。尽管这些肽对结肠的生长促进作用 癌症已被广泛记录在受体的身份和 涉及的细胞内信号通路仍然存在争议。这 研究人员已经鉴定并隔离了新型剪接变体的cDNA G的人类胆囊化蛋白-B/胃蛋白受体（CCK-BR）的成员 蛋白质偶联受体（GPCR）超家族。剪接变体（指定 CCK-BRI4SV用于内含子4包含剪接变体）编码受体蛋白 在其推定的第三个中包含69个其他氨基酸残基 细胞内环域。 CCK-BRI4SV在腺瘤息肉中表达， 结直肠癌，但不在非恶性结肠粘膜中 癌症。表达剪接变体的小鼠BALB3T3细胞表现出 自发，无独立的，振荡性，增加[Ca2+] I 表达野生型CCK-BR（CCK-BRWT）的相同细胞没有。同样，主要 从新鲜切除的结直肠癌分离的人类细胞培养物 表现出[Ca2+]中不依赖配体的，无关的，振荡的增加。两者 BALB3T3和原发性肿瘤细胞，应用G-17（10和200 nm， 分别引起[Ca2+] i的增加。选择性CCK-BR拮抗剂 阻止了G-17刺激的Ca2+响应，但没有自发[Ca2+] I 振荡。除了自发的细胞内信号传导外，BAIB3T3细胞 表达CCK-BRI4SV表现出增加的细胞增殖速率 （大约2.5倍），在没有G-17的情况下，与表达的细胞相比 野生型CCK-BR（CCK-BRWT）。基于这些发现，PI假设 CCK-BRI4SV可以通过两种A调节结直肠癌细胞的生长 胃蛋白独立和依赖性机制，因此起着重要的作用 在结直肠癌中。此外，PI假设 CCK-BRI4SV在结直肠癌生物学中的功能是直接结果 由内含子引起的第三个细胞内环域的结构变化 保留以及这些变化对细胞内信号的影响 转导。为了检查这些假设，他们计划了实验 以下特定目的：1）确定 腺瘤息肉和结肠癌中的CCK-BR剪接变体； 2）到 确定内含子保留对受体介导的细胞内的影响 信号转导和受体脱敏/内在化；和3）到 确定CCK-BR剪接变体的异位表达对 结肠上皮细胞稳态和致癌物诱导的敏感性 使用转基因小鼠模型结肠癌。这些研究将提供 有关新受体剪接的作用的重要和新信息 变体和G-17以及上皮细胞生物学和结肠癌发生的G-Gly。 此外，这些研究将来可能会为 开发用于治疗肽的创新治疗策略 激素敏感的癌症。