Novel Glycoprotein Hormone-Like proteins

描述（由申请人提供）：我们知道有4种糖蛋白激素（LH、FSH、CG和TSH），并且知道它们与属于G蛋白偶联受体（GPCR）超家族的特异性受体相互作用。与其他GPCR相比，糖蛋白激素受体的显著特征是它们含有一个大的富含亮氨酸的含重复序列的胞外结构域。最近，已经克隆了另外五种含有大的富含亮氨酸重复序列的胞外域的GPCR（LGR 4 -8）。这些GPCR与LH/FSH/TSH受体顺序相关，但不结合这些激素。因此，它们属于“孤儿”GPCR类，即等待与配体匹配的受体。我们假设，这些受体结合迄今未描述的蛋白质激素，并提出分离和表征这些激素。我们已经开发了一种策略，使我们能够发现孤儿GPCR的天然配体。简而言之，孤儿GPCR诱导的第二信使反应用于监测从组织提取物中鉴定和纯化该孤儿GPCR配体。我们有初步的数据表明，垂体蛋白提取物在不同阶段的纯化能够引起一个可重复的第二信使反应，在细胞转染LGR 4。最近还通过序列分析发现，人类基因组编码两种蛋白质（α 2和β 10），它们与糖蛋白激素不同，但结构相似。我们建议首先测试α 2和β 10是否可以激活任何孤儿LGRs。我们还建议完成LGR 4的天然配体的分离并确定其序列，以及分离和测序LGR 5和6的天然配体。然后，我们将分析这些新的配体在体外的药理学和生物学活性。最后，我们将研究它们在体内的作用，特别是测试它们是否发挥激素功能。

DESCRIPTION (provided by applicant): We know of 4 glycoprotein hormones (LH, FSH, CG and TSH) and know that they interact with specific receptors that belong to the superfamily of the G protein-coupled receptors (GPCRs). The striking characteristic of the glycoprotein hormone receptors, when compared to other GPCRs, is that they contain a large leucine rich repeat-containing extracellular domain. Recently, five more GPCRs containing large leucine-repeat rich ectodomains have been cloned (LGR4-8). These GPCRs are sequentially-related to LH/FSH/TSH receptors but do not bind these hormones. They fall therefore into the class of the "orphan" GPCRs, receptors awaiting to be matched to a ligand. We hypothesize that these receptors bind thus far undescribed protein hormones and propose to isolate and characterize these hormones. We have developed a strategy that allows us to discover the natural ligands of orphan GPCRs. In short, the orphan GPCR-induced second messenger response is used to monitor the identification and purification of this orphan GPCR ligand from tissue extracts. We have preliminary data that indicate that pituitary protein extracts at various stages of purification are able to elicit a reproducible second messenger response in cells transfected with LGR4. It has also recently been discovered, by sequence analysis, that the human genome encodes two proteins (alpha2 and beta10) that are different but structurally similar to the glycoprotein hormones. We propose first to test whether alpha2 and beta10 can activate any of the orphan LGRs. We propose also to complete the isolation of the natural ligand of LGR4 and determine its sequence and isolate and sequence the natural ligands of LGR5 and 6. We will then analyze these novel ligands for their pharmacological and biological activates in vitro. Finally, we will study their roles in vivo, in particular test whether they exert a hormonal function.