Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology
Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology
复制标题
DOI：
10.1074/jbc.m206931200
复制
发表时间：
2002-10-18
影响因子：
4.8
通讯作者：
Richter, D
中科院分区：
文献类型：
--
作者：
Kreienkamp, HJ;Larusson, HJ;Richter, D
通过结合果蝇基因组数据库搜索和基于逆转录酶 PCR 的 cDNA 分离，克隆了两种 G 蛋白偶联受体，它们是哺乳动物阿片类药物/生长抑素受体最接近的已知无脊椎动物同源物。然而，当通过注射果蝇孤儿受体RNA和共表达的钾通道在非洲爪蟾卵母细胞中功能性表达时，这两种受体都不会被已知的哺乳动物激动剂激活。通过应用反向药理学方法，从成蝇和幼虫的肽提取物中分离出生理配体。纯化配体的 Edman 测序和质谱分析揭示了两种十五肽，它们的不同之处仅在于 N 端焦谷氨酸/谷氨酰胺。这些肽与果蝇基因组数据库的激素前体序列对齐，并且与来自 Manduca sexta 的 Allatostatin C 几乎相同。无论 N 末端修饰如何，两种受体均被合成肽激活。跨膜区 3 中的残基以及跨膜区 6 和 7 之间的环的定点诱变影响配体结合，如先前针对生长抑素受体所述。两个受体基因各自包含三个外显子并以相反方向转录，相距 80 kb，中间没有预测其他基因。受体转录本的定位确定了新的递质系统在视觉信息处理和内分泌调节中的作用。
By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.