Identification of ligand signaling for the stem cell marker LGR5

描述（由申请人提供）：我们鉴定了5个人类LGR受体，它们含有一个大的n端外结构域，含有富含亮氨酸的重复序列，以及7个用于G蛋白偶联的跨膜区域。尽管LGR4/5/6的配体尚不清楚，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发挥了重要作用。这些受体位于肿瘤发生所必需的Wnt信号通路的下游，在多种人类肿瘤中过表达。最近对肠隐窝底部表达lgr5的细胞中一个可诱导的报告基因的分析，允许将这些细胞追踪到不同谱系的肠细胞中。发现LGR5是胃肠道、乳腺、毛囊、性腺等来源的成体和肿瘤干细胞的标记物。由于LGR5在干细胞更新和细胞命运决定中的潜在作用，本研究试图确定LGR5的配体信号传导机制。我们克隆出了与人类LGR4/5/6同源的果蝇受体，并鉴定出其配体为bursicon，是一种含有两种半胱氨酸结的蛋白burs和pburs的异源二聚体。由于这两个果蝇亚基与7种人类BMP（骨形态发生蛋白）拮抗剂同源，我们假设这种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，以缩小寻找旁分泌LGR5配体的范围。早期对组成活性LGR受体的研究允许阐明其G蛋白伴侣。基于LGR5之间保守的跨膜序列，我们将产生LGR5的功能获得突变体来阐明其信号机制。使用锚定外结构域方法，我们已经生成了糖蛋白激素受体的可溶性外结构域作为配体结合功能拮抗剂。我们建议生成LGR5的外域作为“反向配体”，以方便LGR5配体的识别。基于进化守恒、功能获得突变体和反向配体的组合方法可以阐明LGR5的配体信号机制，从而为未来操纵多种组织和肿瘤中的干细胞命运和更新提供机会。

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.