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

项目总结（摘要）

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.