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: 7510574
• 负责人: AARON JW HSUEH
• 金额: $ 23.7万
• 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/7510574
• 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; Follicle Stimulating Hormone; Future; GTP-Binding Proteins; Generations; Genes; Genus Cola; 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; Public Health; 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; interest; intestinal crypt; mutant; novel; paracrine; receptor; response; 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）：我们确定了五个人体LGR受体，其中含有大型N末端外部域，具有富含亮氨酸的重复序列，以及一个用于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突变体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。这些受体是Wnt信号通路的下游，对于肿瘤发生必不可少的，在各种人类肿瘤中过表达。在肠道隐窝底部表达LGR5的细胞中诱导记者基因的最新分析使这些细胞将这些细胞追溯到多种谱系的肠细胞中。发现LGR5是胃肠道，乳腺，毛囊，性腺和其他起源的成年和肿瘤干细胞的标记。由于LGR5在干细胞更新和细胞命运确定中的潜在作用，本提案试图鉴定LGR5的配体信号传导机制。我们已经将果蝇受体直接与人类LGR4/5/6结合起来，并将其配体确定为Bursicon，Bursicon是两个含有蛋白质的蛋白质，毛bur和Pburs的异二聚体。由于这两个蝇亚基与七个人类BMP（骨形态发生蛋白）拮抗剂是同源的，因此我们假设在进化过程中的该配体信号系统的保存，并建议研究LGR5和单个BMP拮抗剂的共表达，以缩小对旁氨基LGR5 Ligands的搜索范围。早期关于组成性活性LGR受体的研究允许阐明其G蛋白伴侣。基于LGR之间的保守跨膜序列，我们将生成推定的LGR5功能增益突变体，以阐明其信号传导机制。使用锚定的外生域方法，我们已经产生了糖蛋白激素受体的可溶性外生素作为配体结合功能拮抗剂。我们建议生成LGR5的外生域作为“反向配体”，以促进LGR5配体识别。基于进化保守，功能收益突变体以及反向配体的组合方法可以阐明LGR5的配体信号传导机制，从而为未来对干细胞命运和不同组织和肿瘤更新的操纵提供了机会。 公共卫生相关性：LGR5受体被证明是胃肠道，乳腺，毛囊，性腺和其他起源的成人和肿瘤干细胞的标志物。提出的对LGR5的同源配体的鉴定可以揭示LGR5在干细胞更新和细胞命运确定中的作用，从而为治疗退化性疾病和各种肿瘤的治疗提供了机会。