Identification of ligand signaling for the stem cell marker LGR5

干细胞标记物 LGR5 配体信号传导的鉴定

• 批准号: 7632206
• 负责人: AARON JW HSUEH
• 金额: $ 19.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7632206
• 关键词: Adult; Affinity Chromatography; Animal Model; Binding; Binding Proteins; Bone Morphogenetic Proteins; Cell Fate Control; Cell Line; Cell surface; Cells; Colon; Coupling; Cystine; Degenerative Disorder; Derivation procedure; Development; Drosophila genus; Embryo; Evolution; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Future; GTP-Binding Proteins; Generations; Genes; Glycoproteins; Gonadal structure; Growth; Hair follicle structure; Hormone Receptor; Hormones; Human; Human Genome; In Situ Hybridization; Individual; Intestines; Investigation; Knockout Mice; Leucine-Rich Repeat; Ligand Binding; Ligands; Mammary gland; Mutant Strains Mice; N-terminal; Names; National Institute of Child Health and Human Development; National Institute of Diabetes and Digestive and Kidney Diseases; Neonatal; Orthologous Gene; Pathway interactions; Phenotype; Property; Proteins; Recombinants; Relaxin; Reporter Genes; Research Project Grants; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Homology; Signal Pathway; Signal Transduction; Stem Cell Development; Stem cells; Stomach; System; Testing; Tissues; Transmembrane Domain; Tumor Stem Cells; Tumor Tissue; base; bursicon; cancer stem cell; design; fetal; fly; gain of function; gain of function mutation; gastrointestinal; genome sequencing; interest; intestinal crypt; mutant; novel; paracrine; public health relevance; receptor; response; stem cell division; stem cell fate; tumor; tumorigenesis

DESCRIPTION (provided by applicant): We identified five human LGR receptors containing a large N-terminal ectodomain with leucine-rich repeats together with a seven transmembrane region for G protein coupling. Although ligands for LGR4/5/6 are still unknown, investigation of LGR4 and LGR5 mutant mice indicated the essential roles of these genes during fetal and neonatal development. These receptors are downstream of the Wnt signaling pathway essential for tumorigenesis and are over-expressed in diverse human tumors. Recent analyses of an inducible reporter gene in LGR5-expressing cells at the base of intestinal crypts allowed the tracing of these cells into intestinal cells of diverse lineages. LGR5 was found to be a marker for adult and tumor stem cells of gastrointestinal, mammary gland, hair follicle, gonadal and other origins. Due to the potential roles of LGR5 in stem cell renewal and cell fate determination, the present proposal attempts to identify the ligand signaling mechanism for LGR5. We have cloned the Drosophila receptor orthologous to human LGR4/5/6 and identified its ligand as bursicon, a heterodimer of two cystine-knot containing proteins, burs and pburs. Because these two fly subunits are homologous to seven human BMP (bone morphogenetic protein) antagonists, we hypothesize the conservation of this ligand signaling system during evolution and propose to investigate the co-expression of LGR5 and individual BMP antagonists to narrow down the search for paracrine LGR5 ligands. Earlier studies on constitutively active LGR receptors allowed the elucidation of their G protein partners. Based on the conserved transmembrane sequences among LGRs, we will generate putative gain-of-function mutants of LGR5 to elucidate its signaling mechanisms. Using an anchored ectodomain approach, we have generated soluble ectodomains of glycoprotein hormone receptors as ligand-binding functional antagonists. We propose to generate the ectodomain of LGR5 for use as a "reverse ligand" to facilitate LGR5 ligand identification. The combined approaches based on evolutionary conservation, gain-of-function mutants, and the reverse ligand could allow the elucidation of the ligand signaling mechanisms for LGR5, thus providing opportunities for future manipulation of stem cell fate and renewal in diverse tissues and tumors. PUBLIC HEALTH RELEVANCE: The LGR5 receptor is shown to be a marker for adult and tumor stem cells of gastrointestinal, mammary gland, hair follicle, gonadal and other origins. Proposed identification of cognate ligands for LGR5 could reveal the role of LGR5 in stem cell renewal and cell fate determination, thus providing opportunities for the management of degenerative diseases and treatment of various tumors.

描述（由申请人提供）：我们鉴定了五种人类 LGR 受体，其中包含具有富含亮氨酸重复序列的大 N 末端胞外域以及用于 G 蛋白偶联的七个跨膜区域。尽管 LGR4/5/6 的配体仍然未知，但对 LGR4 和 LGR5 突变小鼠的研究表明这些基因在胎儿和新生儿发育过程中的重要作用。这些受体位于肿瘤发生所必需的 Wnt 信号通路的下游，并在多种人类肿瘤中过度表达。最近对肠隐窝底部表达 LGR5 的细胞中诱导型报告基因的分析允许将这些细胞追踪到不同谱系的肠细胞。 LGR5被发现是胃肠道、乳腺、毛囊、性腺和其他来源的成人和肿瘤干细胞的标记。由于 LGR5 在干细胞更新和细胞命运决定中的潜在作用，本提案试图鉴定 LGR5 的配体信号传导机制。我们克隆了与人 LGR4/5/6 直系同源的果蝇受体，并鉴定其配体为 bursicon，这是两个含有胱氨酸结的蛋白质 burs 和 pburs 的异二聚体。由于这两个果蝇亚基与七种人类 BMP（骨形态发生蛋白）拮抗剂同源，因此我们假设该配体信号系统在进化过程中是保守的，并建议研究 LGR5 和单个 BMP 拮抗剂的共表达，以缩小旁分泌的搜索范围LGR5 配体。早期对组成型活性 LGR 受体的研究阐明了其 G 蛋白伴侣。基于LGR之间保守的跨膜序列，我们将产生假定的LGR5功能获得突变体以阐明其信号传导机制。使用锚定胞外域方法，我们生成了糖蛋白激素受体的可溶性胞外域作为配体结合功能拮抗剂。我们建议生成 LGR5 的胞外域用作“反向配体”以促进 LGR5 配体识别。基于进化保守、功能获得突变体和反向配体的组合方法可以阐明LGR5的配体信号传导机制，从而为未来操纵不同组织和肿瘤中的干细胞命运和更新提供机会。 公共健康相关性：LGR5 受体被证明是胃肠道、乳腺、毛囊、性腺和其他来源的成人和肿瘤干细胞的标志物。拟议鉴定 LGR5 的同源配体可以揭示 LGR5 在干细胞更新和细胞命运决定中的作用，从而为退行性疾病的管理和各种肿瘤的治疗提供机会。