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: 10380585
• 负责人: Jessica Scales
• 金额: $ 4.68万
• 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 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380585
• 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; GTP-Binding Protein alpha Subunits, Gs; 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; Pharmacology; 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; base; cell motility; cysteinyl leukotriene receptor 2; effective therapy; extracellular; human migration; immunogenic; inhibitor; melanocyte; melanoma; migration; novel; 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） 黑素细胞由一组异质的神经嵴衍生细胞组成，存在于皮肤、眼睛、 耳朵、心脏和大脑。虽然非皮肤部位的黑素细胞功能仍然很差 据了解，皮肤中黑色素细胞的主要功能是合成黑色素，负责 色素沉着并保护皮肤免受紫外线辐射引起的损伤。这里，黑素细胞是 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。许多黑素细胞功能是由 G 蛋白偶联受体介导的 （GPCR），尽管它们在黑色素瘤中发挥作用，但其中大多数尚未在黑色素细胞中得到表征 发病。半胱氨酰白三烯受体 2 (CysLT2R) 是人类黑色素细胞中一种未表征的 GPCR 是葡萄膜黑色素瘤 (UM) 中的一种新型 GPCR 致癌基因，该基因源自葡萄膜黑色素细胞的转化 眼睛。虽然 UM 在人类癌症中很少见，但它是最常见的眼癌，超过 50% 的患者会患上眼癌。 发展致命的转移性疾病。超过 95% 的 UM 具有相互排斥的激活突变 CysLT2R、GNAQ 或 GNA11（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 Ca2+ 通量。这表明 CysLT2R 可以在上游发挥作用 黑素细胞中 Gαq/11 的表达，以及 CysLT2R-Gαq/11-PLCβ4 信号通路的组成型激活可能 是UM细胞高迁移潜力的基础。该提案的总体目标是定义 CysLT2R 介导的信号传导导致人原代黑素细胞细胞迁移的机制。我的 初步研究表明，STIM/ORAI 通道通过 Ca2+ 流入激活钙池操作的 Ca2+ 进入可能 是允许黑素细胞迁移的 CysLT2R 信号传导的关键组成部分，并且转录共同 激活剂 YAP 和 TAZ 的激活导致 UM 细胞迁移，可能介导 CysLT2R 依赖性 黑素细胞迁移。 Thus, I will test my central hypothesis that CysLT2R activation leads to a rapid elevation 通过 STIM/ORAI 通道进入细胞内 Ca2+，从而增强黑色素细胞向下游的迁移 YAP/TAZ 靶基因的转录。目标 1 将采用活体单细胞 Ca2+ 成像和体外迁移 在人黑素细胞和 UM 细胞中进行测定，以确定 STIM/ORAI 介导的 Ca2+ 流入对 CysLT2R 介导的信号传导和细胞迁移。目标 2 将采用生化技术和体外 人类黑素细胞和 UM 细胞的迁移测定，以确定 CysLT2R 如何调节黑素细胞迁移 通过 YAP/TAZ。总的来说，这些数据将定义指导 CysLT2R 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新特征来显着影响人类健康 UM 治疗的目标。布朗大学卓越的研究环境和提供的培训 我的赞助商和共同赞助商将指导我成功完成拟议的研究。