G Protein-Coupled Receptors with Leucine-Rich Repeats

描述(申请人提供)：G蛋白偶联受体超家族 代表了脊椎动物中最大的基因家族之一。在这群人中 蛋白质，促甲状腺激素和促性腺激素受体代表一个亚家族 分别在甲状腺和性腺中起重要作用。这些糖蛋白 激素受体的独特之处在于含有丰富的亮氨酸重复序列 负责配体结合的胞外结构域。基因组计划的进展已经 使得许多新基因的发现成为可能。基于序列相似性 搜索，我们已经确定了五个编码人类的基因，一个线虫和四个 苍蝇孤儿受体分享保守的结构域排列 哺乳动物糖蛋白激素受体，命名为LGRS(富含亮氨酸) 含有重复序列，G蛋白偶联受体)。哺乳动物的孤儿LGRS可以 分为两个亚组，我们的研究集中在一个原型成员上， LGR4。我们建议描述卵巢的表达模式和假定的 这一新型受体的配体以了解其生理功能。 因为糖蛋白激素受体是为数不多的GPCR之一 配体结合区位于胞外结构域，富含亮氨酸的区域重复 同源的LGR胞外区很可能是 这些孤儿LGR。我们将生成LGR4的可溶性胞外结构域 其假定配体的表征(S)。An的初步表征 LGR4胞外结构域剪接变异体允许产生大量的 这个可溶的配基结合区。我们已经提纯了毫克量的 编码假定配体结合区的LGR4剪接变异体和 产生的针对该蛋白的抗体用于免疫细胞化学定位 LGR4在不同卵巢细胞中的表达。Northern杂交分析也证明了 LGR4mRNAs在卵巢中的表达及促性腺激素调节这个 LGR4的可溶性配体结合区将被用作 内源性激动剂的鉴定和亲和纯化 对于这种孤儿受体，我们的初步数据表明，联合治疗 用该蛋白减弱促性腺激素对卵巢重量的刺激 在体内获得。我们建议建立一种基于颗粒细胞的生物检测方法， 用亲和纯化的方法鉴定LGR4的配体(S) 卵泡液和卵巢提取物在这一试点应用中。最终 纯化LGR4LGR4的配体(S)可以提供对其 在卵巢和其他组织中的生理作用。

DESCRIPTION (provided by applicant): The G protein-coupled receptor superfamily represents one of the largest gene families in vertebrates. Among this group of proteins, the thyrotropin and gonadotropin receptors represent a subfamily with important functions in thyroid and gonads, respectively. These glycoprotein hormone receptors are unique in having leucine-rich repeat-containing ectodomains responsible for ligand binding. Advances in genome projects have allowed the discovery of many novel genes. Based on sequence similarity searches, we have identified genes encoding five human, one nematode, and four fly orphan receptors sharing the conserved domain arrangement found in mammalian glycoprotein hormone receptors and named them as LGRS (Leucine-rich repeat containing, G protein-coupled Receptors). The mammalian orphan LGRS can be divided into two subgroups and our studies focus on one prototypic member, LGR4. We propose to characterize the ovarian expression pattern and putative ligands of this novel receptor to understand its physiological functions. Because glycoprotein hormone receptors are among the few GPCRs in which the ligand-binding region resides in the ectodomain, the leucine-rich repeats in the homologous LGR ectodomains are likely to be the ligand-binding regions of these orphan LGRs. We will generate the soluble ectodomain of LGR4 for the characterization of its putative ligand(s). Preliminary characterization of an LGR4 ectodomain-splicing variant allowed the generation of large amounts of this soluble ligand-binding region. We have already purified mg quantities of the LGR4 splicing variant encoding the putative ligand-binding region and generated antibodies against this protein for immunocytochemical localization of LGR4 in different ovarian cells. Northern blot analysis also demonstrated the expression and gonadotropin regulation of LGR4 mRNAs in the ovary. The soluble ligand-binding region for LGR4 will be used as a "reverse ligand" for the identification and affinity purification of putative endogenous agonists for this orphan receptor and our preliminary data indicate that co-treatment with this protein attenuated the gonadotropin stimulation of ovarian weight gain in vivo. We propose to set up a granulosa cell-based bioassay, coupled with affinity purification, to characterize the ligand(s) for LGR4 in follicular fluid and ovarian extracts in this pilot application. Eventual purification of the ligand(s) for LGR4 could provide understanding of its physiological roles in ovary and other tissues.