Growth Hormone Receptor Dimerization & Disulfide Linkage

描述(申请人提供)：生长激素(GH)，主要来自垂体前叶，以内分泌方式调节脊椎动物出生后的生长和新陈代谢。最近的研究还表明，自分泌的生长激素和完整的生长激素轴在动物癌症的形成和行为中发挥了作用。GH受体(GHR)是一个细胞表面糖蛋白细胞因子受体超家族成员，它结合GH在其胞外区(ECD)，并通过其胞内区(ICD)与JAK2酪氨酸激酶的相互作用来激活信号。我们最近对调节GHR可获得性和激活的机制的研究揭示了令人兴奋的见解，包括：1)JAK2关联影响GHR的表面呈现、稳定性和运输；2)降低人T47D乳腺癌细胞中催乳素受体(PRLR)的水平增加GHR的丰度和GH的敏感性；3)新型的构象敏感的抗GHR ECD抗体可能是有用的GH拮抗剂。我们假设：1)JAK2表达水平和PRLR表达水平强烈影响GH的反应性；2)GH诱导的GHR构象变化是GH信号转导的潜在靶点。我们的具体目标是：1.确定JAK2和PRLR调节GHR加工、细胞表面稳定性和下调的机制。我们将研究GHR预聚如何影响JAK2对GHR贩运的S调控，以及GHR ICD酪氨酸残基在GH非依赖和GH依赖的GHR贩运中的作用。PRLR的表达对GHR的可用性、GHR-JAK2关联和GH在乳腺癌细胞中的作用的影响将被检测。2.测定构象特异性抑制性抗GHR ECD单抗的体内效力。我们将在信号研究中表征两个抗GHR抗体和Fab片段的抑制特性，并评估PRLR表达的影响。我们将确定这些试剂在体内对抗GH信号和肿瘤外植体生长的有效性。这些研究探索了生长激素敏感性的重要决定因素。完成后将揭示调节细胞表面生长激素受体可用性和命运的机制，以及调节生长激素敏感性的治疗相关工具。 公共卫生相关性：生长激素是生长和新陈代谢的关键调节因素，最近的研究表明，拮抗生长激素的作用可能在某些癌症中具有治疗潜力。这些生长激素受体的研究在细胞水平上探索生长激素敏感性的决定因素。作为潜在治疗手段的抑制性抗GH受体单抗的获得和发展，可能对我们理解正常生理和癌症治疗具有广泛的相关性。

DESCRIPTION (provided by applicant): Growth hormone (GH), derived largely from the anterior pituitary, regulates postnatal growth and metabolism in vertebrates in an endocrine fashion. Recent studies also suggest roles for autocrine-derived GH and for an intact GH axis in formation and behavior of cancers in animals. GH receptor (GHR) is a cell surface glycoprotein cytokine receptor superfamily member that binds GH in its extracellular domain (ECD) and activates signaling via its intracellular domain's (ICD) interaction with the JAK2 tyrosine kinase. Our recent studies of mechanisms regulating GHR availability and activation reveal exciting insights, including: 1) JAK2 association influences GHR surface presentation, stability, and trafficking; 2) reducing prolactin receptor (PRLR) levels in human T47D breast cancer cells augments GHR abundance and GH sensitivity; 3) novel, conformationally-sensitive anti-GHR ECD antibodies may be useful GH antagonists. We hypothesize: 1) JAK2 expression levels and PRLR expression levels strongly influence GH responsiveness; 2) GH-induced GHR conformational changes that underlie GH signaling are potential targets for therapeutic intervention. Our specific aims are: 1. Determine mechanisms by which JAK2 and PRLR regulate GHR processing, cel surface stability, and downregulation. We will study how GHR predimerization impacts JAK2's modulation of GHR trafficking and the role of GHR ICD tyrosine residues on GH-independent and GH-dependent GHR trafficking. Effects of PRLR expression on GHR availability, GHR-JAK2 association, and GH actions in breast cancer cells will be examined. 2. Determine in vivo efficacy of conformation-specific inhibitory anti-GHR ECD monoclonal antibodies. We will characterize inhibitory properties of two anti-GHR antibodies and Fab fragments in signaling studies, also assessing impact of PRLR expression. We will determine in vivo efficacy of these reagents to antagonize GH signaling and cancer explant growth. These studies probe important determinants of GH sensitivity. Completion will reveal mechanisms regulating cell surface GHR availability and fate and therapeutically relevant tools to modulate GH sensitivity. PUBLIC HEALTH RELEVANCE: Growth hormone is a key regulator of growth and metabolism and recent work suggests that antagonism of growth hormone action may be of therapeutic potential in certain cancers. These studies of the growth hormone receptor investigate the determinants of growth hormone sensitivity at the cellular level. The knowledge gained and the development of inhibitory anti-GH receptor monoclonal antibodies as potential therapeutics may have broad relevance in our understanding of normal physiology and in treatment of cancer.