CHARACTERIZATION OF GONADAL RECEPTORS AND GONADOTROPIN BIOLOGICAL ACTIVITY

对LH受体基因组结构的分析显示， 跨越至少75 kb，并在其内部含有两个潜在的启动子区域， 5'非编码区的前2kb，具有CAT样基序的TATA盒 （-1694）和SP1结合位点区域（-83、-103和-143）。 编码 LH受体基因的一个区域由11个外显子组成， 内含子，所有位于推定的细胞外结构域之前， 第一跨膜区。 外显子1-10编码细胞外区域 和外显子11用于跨膜和胞质结构域。 几 鉴定了LH受体的替代截短形式， 由完全（外显子9）或部分（外显子11）外显子缺失引起 包含在基因组结构中。 外显子2-8近似区域 LH、TSH和LH中存在的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），以及跨膜结构域和G蛋白 偶联元件（外显子11）。 外显子9可能是核心的重要性， 激素结合结构域的构型，并且也可以作为激素结合结构域的一种结构域发挥功能。 激素或受体的碳水化合物结合结构域。 的 促性腺激素结合结构域位于细胞外区， 受体，并可能涵盖跨越外显子1-10的区域， 由折叠和二硫环组成的重要三级结构。 单外显子（外显子11）可能是由“外显子改组”机制引起的 在进化过程中或将一个完整的功能域引入 基因的编码区。 hCG结合的主要减少 去糖基化纯化的受体表明N-连接的碳水化合物 成熟受体上的一个或多个链对于最佳的受体结合是必需的。 约束力 Asn 173转化为Gln导致完全丧失了 激素结合活性，并在此去除碳水化合物链 位置可能是负责所观察到的取消激素 纯化的受体在去糖基化后的结合活性。 突变体 含Asn 77 Gln和Asn 152 Gln的蛋白质与 细胞受体总数减少（80%）。 部分保留 这些突变体的活性表明， 链不是必需的，或者其它糖基化链可以部分地 补偿其功能。 其余的突变受体 潜在的糖基化位点（269、277和291）在 约束力 在激素结合活性的变化后消除 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糖链 受体的

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.