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（申请人摘要）：结直肠癌是第三大癌症 在美国，结肠癌的发生是一个复杂的癌症死亡原因， 涉及信号通路渐进变化的多步骤过程 调节肠上皮细胞增殖、分化 程序化死亡。肽类激素胃泌素 1-17 (G-17) 及其 非酰胺化前体，甘氨酸延长胃泌素 (G-Gly)，发挥有效的营养作用 对结肠癌细胞的影响。长期目标是了解 这些肽激素在上皮细胞生物学和结肠的调节中 致癌作用。虽然这些肽对结肠有促进生长的作用 癌症已被广泛记录，受体的身份和 所涉及的细胞内信号通路仍存在争议。这 研究人员已经鉴定并分离出一种新的剪接变体的 cDNA 人类胆囊收缩素-B/胃泌素受体 (CCK-BR)，G 的成员 蛋白质偶联受体（GPCR）超家族。剪接变体（指定 CCK-BRi4sv（含有剪接变体的内含子 4）编码受体蛋白 在其推定的第三个氨基酸残基中含有 69 个额外的氨基酸残基 细胞内环结构域。 CCK-BRi4sv 在腺瘤性息肉中表达 结直肠癌，但不包括邻近结直肠癌的非恶性结肠粘膜 癌症。表达剪接变体的小鼠 Balb3T3 细胞 自发的、不依赖配体的、振荡的、[Ca2+]i 增加，而 表达野生型 CCK-BR (CCK-BRwt) 的相同细胞却没有。同样，初级 从刚切除的结直肠癌中分离出的人类细胞培养物 表现出不依赖配体的[Ca2+]振荡增加。对于两者 Balb3T3 和原发性肿瘤细胞，G-17（10 和 200 nM， 分别)导致[Ca2+]i增加。选择性 CCK-BR 拮抗剂 阻断 G-17 刺激的 Ca2+ 反应，但不阻断自发的 [Ca2+]i 振荡。除了自发的细胞内信号传导外，BaIb3T3 细胞 表达 CCK-BRi4sv 表现出细胞增殖率增加 与表达 G-17 的细胞相比（约 2.5 倍） 野生型 CCK-BR (CCK-BRwt)。根据这些发现，PI 假设 CCK-BRi4sv 可能通过以下两种方式调节结直肠癌细胞的生长： 胃泌素独立和依赖机制因而发挥重要作用 在结直肠癌发生过程中。此外，PI 假设 CCK-BRi4sv 在结直肠癌生物学中的功能是以下因素的直接结果 由内含子引起的第三细胞内环结构域的结构变化 保留，以及这些变化对细胞内信号的影响 转导。为了检验这些假设，他们计划进行实验 以下具体目标：1）确定空间和时间表达 腺瘤性息肉和结肠癌中的 CCK-BR 剪接变异体； 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/内化； 3) 至 确定 CCK-BR 剪接变体的异位表达对 结肠上皮细胞稳态和对致癌物质的易感性 使用转基因小鼠模型进行结肠癌。这些研究将提供 关于新型受体剪接作用的重要新信息 变体以及 G-17 和 G-Gly 在上皮细胞生物学和结肠癌发生中的作用。 此外，这些研究可能为未来的研究提供基础。 开发肽治疗的创新治疗策略 激素敏感性癌症。