Stability of prolactin receptor and prolactin signaling

描述（申请人提供）：垂体激素催乳素（PRL）通过促乳素受体（PRLr）起作用，促进乳腺上皮细胞的生长和存活，在乳腺肿瘤发生中起关键作用。PRL信号是通过PRLr对配体的下调来控制的，其机制在很大程度上是未知的。我们的初步数据表明(i) bTrCP E3泛素连接酶通过PRLr泛素化和依赖于PRLr Ser349磷酸化的溶酶体降解限制了PRL信号传导的程度，（ii）糖原合成酶激酶3 （GSK3）与PRLr Ser349磷酸化有关，（iii） Ser349磷酸化以及PRLr-bTrCP相互作用和PRLr的蛋白水解在乳腺癌细胞中受损。我们假设，PRLr在Ser349上的gsk3依赖性磷酸化导致bTrCP的募集，随后是PRLr的泛素化、内吞/分选和溶酶体降解，从而限制了细胞对PRL的反应程度。在乳腺癌中，致癌信号诱导的PRLr磷酸化和转换异常可能会增加PRL信号传导的程度，促进PRL介导的细胞生长和存活。为了验证这些假设，我们提出：(1)确定PRLr稳定性在可能表现出PRLr Ser349磷酸化受损的乳腺癌细胞和组织中是如何调节的；(2)明确PRLr内吞（内化和后内化转运）所需的基序，以及btrcp依赖性泛素化在这些过程中的作用；(3)确定GSK3是否通过磷酸化PRLr的Ser349位点，参与PRLr的泛素化和降解，以及调控PRL信号传导的程度；(4)确定PRLr的稳定是否会增加体外和小鼠乳腺细胞对PRL的反应程度。意义：这些研究对我们了解乳腺癌发生的事件很重要，因此，与NCI的使命相关。完成这些实验将有可能：(i)确定PRLr稳定是乳腺肿瘤发生中的一个新事件；(2)深入了解激素受体下调的机制，扩大我们对PRL细胞反应调控机制的认识；(3)阐明PRLr稳定性在乳腺癌发病机制中的作用，为改变PRL依赖性乳腺癌细胞的存活水平提供新的手段。

DESCRIPTION (provided by applicant): Pituitary hormone prolactin (PRL), which promotes growth and survival of mammary epithelial cells and plays a key role in breast tumorigenesis, acts through PRL receptor (PRLr). PRL signaling is controlled via down regulation of PRLr in response to the ligand via the mechanisms that are yet largely unknown. Our preliminary data suggest that (i) the bTrCP E3 ubiquitin ligase limits the extent of PRL signaling via PRLr ubiquitination and lysosomal degradation that depends on PRLr phosphorylation on Ser349, (ii) glycogen synthase kinase 3 (GSK3) is implicated in PRLr phosphorylation on Ser349, and (iii) this Ser349 phosphorylation as well as PRLr-bTrCP interaction and the proteolysis of PRLr are impaired in breast cancer cells. We hypothesize that GSK3-dependent phosphorylation of PRLr on Ser349 leads to the recruitment of bTrCP followed by ubiquitination, endocytosis/sorting and lysosomal degradation of PRLr to limit the extent of cellular responses to PRL. In breast cancers, oncogenic signaling-induced aberrations in PRLr phosphorylation and turnover might augment the extent of PRL signaling and promote PRL-mediated cell growth and survival. To test these hypotheses, we propose to: (1) Determine how PRLr stability is regulated in breast cancer cells and tissues that may exhibit impaired PRLr phosphorylation on Ser349; (2) Define the motifs required for PRLr endocytosis (internalization and post-internalization trafficking) and the role of bTrcp-dependent ubiquitination in these processes; (3) Determine whether GSK3, via its ability to phosphorylate PRLr on Ser349, plays a role in ubiquitination and degradation of PRLr and in the regulation of the extent of PRL signaling; (4) Determine whether stabilization of PRLr results in augmentation of the extent of responses to PRL in breast cells in vitro and in mice. Significance: these studies are important for our understanding of the events that occur in breast cancers and, therefore, are relevant to the mission of NCI. Completion of these experiments will potentially: (i). identify PRLr stabilization as a novel event in breast tumorigenesis; (ii) gain insight in the mechanisms underlying down regulation of hormone receptors and expand our knowledge on the mechanisms regulating the cellular responses to PRL, (iii) shed light on the role of PRLr stability in pathogenesis of breast cancer and offer new means for altering the levels of PRL-dependent survival of breast cancer cells.