Novel Glycoprotein Hormone-Like proteins

描述(申请人提供)：我们知道4种糖蛋白激素(黄体生成素、卵泡刺激素、卵泡刺激素和促甲状腺激素)，并知道它们与属于G蛋白偶联受体(GPCRs)超家族的特定受体相互作用。与其他GPCRs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重复序列的胞外结构域。最近又克隆了5个含有大的富含亮氨酸重复序列的胞外区的GPCRs(LGR4-8)。这些GPCRs与黄体生成素/卵泡刺激素/促甲状腺激素受体顺序相关，但不与这些激素结合。因此，它们属于“孤儿”GPCRs的类别，即等待与配体配对的受体。我们假设这些受体结合了迄今为止未被描述的蛋白质激素，并建议分离和鉴定这些激素。我们已经开发了一种策略，使我们能够发现孤儿GPCRs的天然配体。简而言之，孤儿GPCR诱导的第二信使反应被用来监测从组织提取液中鉴定和纯化这种孤儿GPCRL。我们有初步数据表明，不同纯化阶段的垂体蛋白提取物能够在转LGR4的细胞中诱发可重复的第二信使反应。最近还发现，通过序列分析，人类基因组编码两种蛋白质(α2和β10)，这两种蛋白质与糖蛋白激素不同，但结构相似。我们建议首先测试Alpha2和Beta10是否可以激活任何孤儿LGR。我们还建议完成LGR4天然配体的分离和序列测定，并分离和测序LGR5和6的天然配体。然后我们将在体外分析这些新的配体的药理和生物活性。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.