Identification of ligand signaling for the stem cell marker LGR5

描述（由申请人提供）：我们鉴定了五种人LGR受体，其含有具有富含亮氨酸重复序列的大N端胞外域以及用于G蛋白偶联的七个跨膜区。虽然LGR 4/5/6的配体仍然是未知的，但对LGR 4和LGR 5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。这些受体是肿瘤发生所必需的Wnt信号通路的下游，并且在多种人类肿瘤中过表达。最近的分析LGR 5表达细胞的诱导型报告基因在肠隐窝的基础上允许这些细胞的跟踪到不同谱系的肠细胞。发现LGR 5是胃肠、乳腺、毛囊、性腺和其他来源的成体和肿瘤干细胞的标志物。由于LGR 5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LGR 5的配体信号传导机制。我们已经克隆了与人类LGR 4/5/6直系同源的果蝇受体，并鉴定其配体为囊腺激素（bursicon），这是两种含胱氨酸结的蛋白质（布尔斯和p布尔斯）的异二聚体。由于这两个苍蝇亚基是同源的七个人骨形态发生蛋白（骨形态发生蛋白）拮抗剂，我们假设在进化过程中保护这个配体信号系统，并建议调查LGR 5和单个BMP拮抗剂的共表达，以缩小寻找旁分泌LGR 5配体。对组成型活性LGR受体的早期研究允许阐明其G蛋白伴侣。基于LGR之间保守的跨膜序列，我们将产生推定的LGR 5的功能获得性突变体，以阐明其信号转导机制。使用锚定胞外域的方法，我们已经产生了可溶性糖蛋白激素受体的胞外域作为配体结合的功能性拮抗剂。我们建议产生LGR 5的胞外域用作“反向配体”以促进LGR 5配体鉴定。基于进化保守性、功能获得性突变体和反向配体的组合方法可以阐明LGR 5的配体信号传导机制，从而为将来操纵干细胞命运和在不同组织和肿瘤中更新提供机会。 公共卫生相关性：LGR 5受体显示为胃肠、乳腺、毛囊、性腺和其它来源的成体和肿瘤干细胞的标志物。提出的LGR 5同源配体的鉴定可以揭示LGR 5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。

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.