CHARACTERIZATION OF GONADAL RECEPTORS AND GONADOTROPIN BIOLOGICAL ACTIVITY

性腺受体和促性腺激素生物活性的表征

• 批准号: 3857059
• 负责人: M L DUFAU
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3857059
• 关键词: G protein; Leydig cells; RNA splicing; affinity chromatography; chemical binding; chorionic gonadotropin; conformation; gel filtration chromatography; genetic promoter element; glycosylation; high performance liquid chromatography; hormone receptor; hormone regulation /control mechanism; laboratory rat; luteinizing hormone; messenger RNA; ovary; pituitary gonadal axis; posttranslational modifications; prolactin; protein sequence; protein structure; protein structure function; receptor coupling

Analysis of the genomic structure of the LH receptor revealed that the gene spans at least 75 kb and contains two potential promoter regions within the first 2kb of the 5' non-coding region, a TATA box with a CAT-like motif (-1694) and a region of SP1 binding sites (-83, -103 and -143). The coding region of the LH receptor gene consists of 11 exons separated by 10 introns, all located within the putative extracellular domain prior to the first transmembrane region. Exons 1-10 code for the extracellular region and exon 11 for the transmembrane and cytoplasmic domains. Several alternate truncated forms of the LH receptor were identified and shown to result from deletions of complete (exon 9) or partial (exon 11) exons contained within the genomic structure. Exons 2-8 approximate regions of the 14 consecutive 20 amino acid repeat motifs present in the LH, TSH and FSH receptors. Exons 1, 9, 10 and 11 were found to contain important structural elements including conserved cysteines (exons 1, 9, 10 and 11), a soybean lectin motif (exon 9), and a transmembrane domain and G-protein coupling elements (exon 11). Exon 9 may be of central importance to the configuration of the hormone binding domain and may also function as a carbohydrate binding domain for either the hormone or the receptor. The gonadotropin binding domain resides in the extracellular region of the receptor and probably encompasses the region spanning exons 1-10 and has significant tertiary structure consisting of folding and disulfide loops. The single exon (exon 11) could result from an "exon shuffling" mechanism during evolution or the introduction of a complete functional domain into the coding region of the gene. The major reduction in hCG binding to the deglycosylated purified receptor indicates that N-linked carbohydrate chain(s) on the mature receptor are necessary for optimal hormone-receptor binding. The conversion of Asn173 to Gln resulted in complete loss of hormone binding activity, and removal of a carbohydrate chain at this position is probably responsible for the observed abolition of hormone binding activity of the purified receptor upon deglycosylation. Mutants containing Asn77Gln and Asn152Gln were associated with significant decreases in the total number of cell receptors (80%). Partial retention of activity by these mutants indicates that either N-linked carbohydrate chains are not essential or other glycosylation chains can partially compensate for their functions. Mutant receptors at the remaining potential glycosylation sites (269, 277 and 291) exhibited no alteration in binding. The changes in hormone binding activity upon elimination of potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.

LH 受体基因组结构分析表明，该基因 跨度至少 75 kb，并且包含两个潜在的启动子区域 5'非编码区的前 2kb，一个带有类似 CAT 基序的 TATA 盒 (-1694) 和 SP1 结合位点区域 (-83、-103 和 -143)。 编码 LH 受体基因区域由 11 个外显子组成，外显子之间间隔 10 内含子，全部位于推定的细胞外结构域内 第一跨膜区。 细胞外区域的外显子 1-10 代码 外显子 11 为跨膜结构域和细胞质结构域。 一些 LH 受体的替代截短形式已被鉴定并显示 完整（外显子 9）或部分（外显子 11）外显子删除的结果 包含在基因组结构中。 外显子 2-8 的大致区域 LH、TSH 和 TSH 中存在 14 个连续的 20 个氨基酸重复基序 FSH 受体。 外显子 1、9、10 和 11 被发现含有重要的 结构元件，包括保守的半胱氨酸（外显子 1、9、10 和 11）， 大豆凝集素基序（外显子 9）、跨膜结构域和 G 蛋白 耦合元件（外显子 11）。 外显子 9 可能对于 激素结合结构域的构型，也可以作为 激素或受体的碳水化合物结合域。 这 促性腺激素结合域位于细胞外区域 受体，可能涵盖跨越外显子 1-10 的区域，并且具有 由折叠环和二硫环组成的重要三级结构。 单个外显子（外显子 11）可能是由“外显子改组”机制产生的 在进化过程中或将完整的功能域引入到 基因的编码区。 hCG 与 hCG 的结合显着减少 去糖基化的纯化受体表明 N-连接碳水化合物 成熟受体上的链对于最佳激素受体是必需的 绑定。 Asn173 转化为 Gln 导致完全丧失 激素结合活性，并去除碳水化合物链 该位置可能是观察到的激素废除的原因 纯化的受体在去糖基化后的结合活性。 突变体 含有 Asn77Gln 和 Asn152Gln 的人与显着相关 细胞受体总数减少（80%）。 部分保留 这些突变体的活性表明，N-连接碳水化合物 链不是必需的，或者其他糖基化链可以部分地 补偿它们的功能。 剩余的突变受体 潜在的糖基化位点（269、277 和 291）没有表现出改变 绑定。 消除后激素结合活性的变化 77、152 和 173 处的潜在糖基化位点表明存在 大鼠卵巢 LH/hCG 中这些位置的功能性碳水化合物链 受体。