Mechanisms of cysteinyl leukotriene receptor 2 (CysLT2R)-mediated signaling and migration in melanocytes and uveal melanoma

半胱氨酰白三烯受体 2 (CysLT2R) 介导的黑素细胞和葡萄膜黑色素瘤中的信号传导和迁移机制

• 批准号: 10608124
• 负责人: Jessica Scales
• 金额: $ 2.27万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10608124
• 关键词: Biochemical; Brain; Cell division; Cell physiology; Cells; Data; Disease; Down-Regulation; Ear; Ensure; Environment; Eye; Family; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GNAQ gene; GTP-Binding Protein alpha Subunits; Gene Silencing; Genes; Genetic Transcription; Goals; Hair; Health; Heart; Heterotrimeric GTP-Binding Proteins; Homeostasis; Human; Image; In Vitro; Knowledge; Lead; Leukotriene C4; Leukotriene D4; Ligands; Link; Location; Malignant Neoplasms; Measures; Mediating; Melanins; Melanoma Cell; Migration Assay; Molecular; Mutation; Neural Crest; Oncogenes; Pathogenesis; Pathologic; Pathway interactions; Patients; Phenotype; Phospholipase C; Physiological; Physiology; Pigmentation Disorders; Pigmentation physiologic function; Pigments; Polygenic Traits; Proteins; Radiation induced damage; Recurrence; Research; Role; Signal Pathway; Signal Transduction; Site; Skin; Skin Pigmentation; Skin injury; Techniques; Testing; Time; Training; Transcription Coactivator; Transcriptional Activation; Transcriptional Coactivator with PDZ-Binding Motif; UV induced; Ultraviolet Rays; Universities; Uveal Melanoma; Vitiligo; candidate identification; cell motility; cysteinyl leukotriene receptor 2; effective therapy; extracellular; human migration; immunogenic; inhibitor; melanocyte; melanoma; migration; novel; pharmacologic; preservation; receptor function; skin barrier; tumor; wound

PROJECT SUMMARY (ABSTRACT) Melanocytes comprise a heterogeneous group of neural crest-derived cells that are present in the skin, eyes, ear, heart, and brain. While the function of melanocytes present in non-cutaneous locations remain poorly understood, a primary function of melanocytes in the skin is to synthesize the melanin pigments responsible for pigmentation and shielding the skin from ultraviolet radiation-induced damage. Here, melanocytes are homogenously distributed to ensure uniform skin pigmentation and will undergo cell division and migration to repopulate sites of skin injury. Many melanocyte functions are mediated by G-protein-coupled receptors (GPCRs), most of which have yet to be characterized in melanocytes despite their role in melanoma pathogenesis. Cysteinyl leukotriene receptor 2 (CysLT2R) is an uncharacterized GPCR in human melanocytes and is a novel GPCR oncogene in uveal melanoma (UM), which arises from transformed melanocytes in the eye. While rare among human cancers, UM is the most common ocular cancer in which >50% of patients will develop fatal metastatic disease. More than 95% of UMs harbor mutually exclusive activating mutations in CysLT2R, GNAQ or GNA11 (G-protein α-subunits of the Gαq/11 family), or PLCβ4 (phospholipase C β4), which is activated downstream of Gαq/11 and stimulates Ca2+ flux. This suggests that CysLT2R could function upstream of Gαq/11 in melanocytes, and the constitutive activation of the CysLT2R-Gαq/11-PLCβ4 signaling pathway may underlie the high migration potential of UM cells. The overarching objective of this proposal is to define the mechanisms of CysLT2R-mediated signaling leading to cellular migration of human primary melanocytes. My preliminary studies show that activation of store-operated Ca2+ entry via Ca2+ influx by STIM/ORAI channels may be a critical component of CysLT2R signaling that permits melanocyte migration, and that the transcription co- activators YAP and TAZ, whose activation leads to UM cellular migration, may mediate CysLT2R-dependent melanocyte migration. Thus, I will test my central hypothesis that CysLT2R activation leads to a rapid elevation in intracellular Ca2+ via STIM/ORAI channels that results in enhanced melanocyte migration through downstream transcription of YAP/TAZ target genes. Aim 1 will employ live single-cell Ca2+ imaging and in vitro migration assays in human melanocytes and UM cells to determine the contribution of STIM/ORAI-mediated Ca2+ influx to CysLT2R-mediated signaling and cellular migration. Aim 2 will employ biochemical techniques and in vitro migration assays in human melanocytes and UM cells to define how CysLT2R regulates melanocyte migration through YAP/TAZ. Collectively, these data will define the mechanisms that direct CysLT2R-mediated signaling and migration in melanocytes, which has the potential to significantly impact human health by identifying novel targets for the treatment of UM. The exceptional research environment at Brown University and training provided by my Sponsor and Co-Sponsor will guide me towards the successful completion of the proposed research.

项目概要（摘要） 黑素细胞包括存在于皮肤、眼睛 耳朵心脏和大脑虽然存在于非皮肤位置的黑素细胞的功能仍然很差， 据了解，皮肤中黑色素细胞的主要功能是合成黑色素， 色素沉着和保护皮肤免受紫外线辐射引起的损伤。在这里，黑素细胞 均匀分布，以确保均匀的皮肤色素沉着，并将经历细胞分裂和迁移， 重新填充皮肤损伤部位。许多黑素细胞的功能是由G蛋白偶联受体介导的 （GPCR），其中大多数尚未在黑素细胞中表征，尽管它们在黑色素瘤中起作用 发病机制半胱氨酰白三烯受体2（CysLT 2 R）是人类黑素细胞中一种未鉴定的GPCR 并且是葡萄膜黑色素瘤（UM）中的一种新的GPCR癌基因，其产生于葡萄膜黑色素瘤中的转化黑素细胞。 眼睛虽然在人类癌症中很少见，但UM是最常见的眼部癌症，其中>50%的患者会 发展成致命的转移性疾病超过95%的UM在以下方面具有互斥的激活突变： CysLT 2 R、GNAQ或GNA 11（Gαq/11家族的G蛋白α亚基）或PLCβ4（磷脂酶C β4）， 在Gαq/11下游被激活，并刺激Ca 2+通量。这表明CysLT 2 R可以在上游发挥作用， CysLT 2 R-G α q/11-PLCβ4信号通路的组成性激活可能与黑素细胞中Gαq/11的表达有关， 是UM细胞高迁移潜力的基础。本提案的总体目标是确定 CysLT 2 R介导的信号传导机制导致人原代黑素细胞的细胞迁移。我 初步研究表明，通过STIM/奥赖通道的Ca 2+内流激活钙库操作的Ca 2+内流可能 是CysLT 2 R信号传导的关键成分，允许黑素细胞迁移，并且转录协同作用是可能的。 激活剂雅普和TAZ，其激活导致UM细胞迁移，可能介导CysLT 2 R依赖性 黑素细胞迁移因此，我将测试我的中心假设，即CysLT 2 R激活导致快速升高 在细胞内Ca 2+通过STIM/奥赖通道，导致增强的黑素细胞迁移通过下游 雅普/TAZ靶基因的转录。目的1将采用活单细胞钙离子成像和体外迁移 在人黑素细胞和UM细胞中测定STIM/ORAI介导的Ca 2+内流对 CysLT 2 R介导的信号传导和细胞迁移。目标2将采用生物化学技术和体外 在人黑素细胞和UM细胞中的迁移测定，以确定CysLT 2 R如何调节黑素细胞迁移 通过雅普/塔兹。总的来说，这些数据将定义指导CysLT 2 R介导的信号传导的机制。 和黑素细胞的迁移，这有可能通过识别新的 治疗UM的目标。布朗大学卓越的研究环境和提供的培训 我的申办者和联合申办者的指导将指导我成功完成拟定的研究。