GRP Receptor Signaling: Role of the C-Terminal Domain

GRP 受体信号转导：C 端结构域的作用

• 批准号: 6917045
• 负责人: GLENN SCOTT KROOG
• 金额: $ 15.93万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917045
• 关键词: arrestins; biological signal transduction; confocal scanning microscopy; cyanogen bromide; enzyme activity; gastrin releasing peptide; immunoprecipitation; mass spectrometry; mitogen activated protein kinase; neuropeptide receptor; phosphorylation; protein protein interaction; protein structure function; protein transport; trypsin; yeast two hybrid system

DESCRIPTION (provided by applicant): Tumorigenesis and/or proliferation involve interactions of neoplastic cells with each other and with non-malignant host cells. Binding of diffusible factors with cognate receptors mediate many of these interactions. Bombesin-like peptides (BLPs) are a family of regulatory neuropeptides. Gastrin-releasing peptide (GRP) is a human BLP that elicits many physiological effects including mitogenesis. GRP induces cell proliferation by binding to a GRP-preferring G-protein coupled receptor (GRPr) and activating several signal transduction cascades. GRPr and other G protein coupled receptors (GPCRs) may be involved in the promotion and proliferation of several malignancies. Therefore, interruption of GRP-dependent signaling offers a potentially powerful approach for treating cancers expressing GRPr. Elucidating molecular pathways involved in GRPr-mediated signaling and growth may uncover additional targets for chemotherapy, or strategies to combine anti-GPCR therapy with other agents. Therefore, we are studying the function and regulation of GRPr. One domain that is likely to be crucial for some aspects of GRPr-mediated signaling is the carboxyl terminal domain (CTD). CTDs of many GPCRs including GRPr contain sites for phosphorylation, and CTD are involved in regulatory processes including internalization and desensitization. Recent data has implicated the CTD of some GPCRs in activation of signaling. Thus, the CTD of GRPr is likely to be required for regulation of GRP signaling. A central goal of this application is to determine how the CTD of GRPr and GRPr phosphorylation affect receptor-mediated signaling and desensitization. Several complementary approaches will be used: (1) functions and regulation of GRPr mutants which are poor substrates for agonist-induced phosphorylation will be studied in a transfected cell system and compared with wildtype GRPr, (2) GRPr phosphorylation sites will be determined using mass spectrometry, and (3) Novel signaling partners for GRPr will be identified and characterized. Candidate GRPr binding proteins have been identified in a yeast two-hybrid screen in which the CTD was used as a bait. The candidate signaling partners will be evaluated using assays that (a) document and analyze GRPr-binding protein interactions, and (b) determine the roles of GRPr binding proteins in signaling in intact cells. Tumor cells expressing endogenous GRPr will be studied to assess the physiological relevance of the results from each Specific Aim.

描述（由申请人提供）： 肿瘤发生和/或增殖涉及肿瘤细胞彼此之间以及与非恶性宿主细胞之间的相互作用。 扩散因子与同源受体的结合介导了许多这些相互作用。 蛙皮素样肽（BLP）是一类调节性神经肽。 胃泌素释放肽（GRP）是一种具有促有丝分裂等多种生理作用的人源性BLP。 GRP通过与GRP偏好的G蛋白偶联受体（GRPr）结合并激活几个信号转导级联来诱导细胞增殖。 GRPr和其他G蛋白偶联受体（GPCR）可能参与几种恶性肿瘤的促进和增殖。 因此，中断GRP依赖性信号传导为治疗表达GRPr的癌症提供了一种潜在的强大方法。 阐明参与GRPr介导的信号传导和生长的分子途径可能会发现化疗的其他靶点，或将联合收割机抗GPCR治疗与其他药物结合的策略。 因此，我们正在研究GRPr的功能和调节。 羧基末端结构域（CTD）可能对GRPr介导的信号传导的某些方面至关重要。 包括GRPr在内的许多GPCR的CTD含有磷酸化位点，并且CTD参与包括内化和脱敏的调节过程。 最近的数据表明，一些GPCR的CTD参与了信号传导的激活。 因此，GRPr的CTD可能是调节GRP信号传导所必需的。 本申请的中心目标是确定GRPr和GRPr磷酸化的CTD如何影响受体介导的信号传导和脱敏。 将使用几种互补的方法：（1）在转染的细胞系统中研究GRPr突变体的功能和调节，这些突变体是激动剂诱导的磷酸化的不良底物，并与野生型GRPr进行比较，（2）使用质谱法确定GRPr磷酸化位点，以及（3）识别和表征GRPr的新信号传导伴侣。 候选GRPr结合蛋白已被确定在酵母双杂交筛选，其中CTD被用作诱饵。 将使用以下测定来评估候选信号伴侣：（a）记录和分析GRPr结合蛋白相互作用，以及（B）确定GRPr结合蛋白在完整细胞中信号传导中的作用。 将研究表达内源性GRPr的肿瘤细胞，以评估每个特定目的结果的生理相关性。