Stability of prolactin receptor and prolactin signaling

描述(申请人提供)：垂体激素催乳素(PRL)，促进乳腺上皮细胞的生长和存活，在乳腺肿瘤的发生中起关键作用，通过PRL受体(PRLr)起作用。PRL信号是通过下调PRLr对配体的反应来控制的，其机制目前尚不清楚。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Ser349上的PRLr磷酸化的溶酶体降解来限制PRL信号的程度，(Ii)糖原合成酶激酶3(GSK3)与Ser349上的PRLr磷酸化有关，(Iii)这种Ser349磷酸化以及PRLr-bTrCP相互作用和PRLR的蛋白分解在乳腺癌细胞中受到损害。我们推测，依赖于GSK3的PRLr在Ser349上的磷酸化导致bTrCP的募集，随后是PRLr的泛素化、内吞/分选和溶酶体降解，从而限制细胞对PRL的反应程度。在乳腺癌中，癌基因信号诱导的PRLr磷酸化和更新的异常可能增加PRL信号的程度，促进PRL介导的细胞生长和存活。为了验证这些假说，我们建议：(1)确定乳癌细胞和可能在Ser349上表现出PRLr磷酸化受损的组织中PRLr的稳定性是如何调节的；(2)确定PRLr内吞所需的基序(内化和内化后转运)以及bTrcp依赖的泛素化在这些过程中的作用；(3)确定GSK3是否通过其在Ser349上磷酸化PRLr的能力，在PRLR的泛素化和降解以及PRL信号的调节范围中发挥作用；(4)确定PRLr的稳定是否导致体外和小鼠乳腺细胞对PRL的反应程度增加。意义：这些研究对于我们理解发生在乳腺癌中的事件很重要，因此与NCI的使命相关。这些实验的完成可能会：(I)。确定PRLr稳定是乳腺肿瘤发生中的一个新事件；(Ii)深入了解激素受体下调的机制，并扩大我们对调节细胞对PRL反应的机制的了解；(Iii)阐明PRLr稳定在乳腺癌发病机制中的作用，并为改变乳腺癌细胞PRL依赖的生存水平提供新的手段。

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.