University of Victoria, Department of Biology, Victoria, British Columbia, Canada V8W 3N5; Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037, USA.
Integr Comp Biol. 2008 Nov;48(5):570-87. doi: 10.1093/icb/icn070. Epub 2008 Jul 21.
Reproduction in all vertebrates requires the brain hormone gonadotropin-releasing hormone (GnRH) to activate a cascade of events leading to gametogenesis. All vertebrates studied to date have one to three forms of GnRH in specific but different neurons in the brain. In addition, at least one type of GnRH receptor is present in each vertebrate for activation of specific physiological events within a target cell. Humans possess two types of GnRH (GnRH1 and GnRH2) but only one functional GnRH receptor. Zebrafish, Danio rerio, also have two types of GnRH (GnRH2 and GnRH3), although in contrast to humans, zebrafish appear to have four different GnRH receptors in their genome. To characterize the biological significance of multiple GnRH receptors within a single species, we cloned four GnRH receptor cDNAs from zebrafish and compared their structures, expression, and cell physiology. The zebrafish receptors are 7-transmembrane G-protein coupled receptors with amino-acid sequence identities ranging from 45 to 71% among the four receptors. High sequence similarity was observed among the seven helices of zebrafish GnRHRs compared with the human GnRHR, the green monkey type II GnRHR, and the two goldfish GnRHRs. Also, key amino acids for putative ligand binding, disulfide bond formation, N-glycosylation, and G-protein coupling were present in the extracellular and intracellular domains. The four zebrafish receptors were expressed in a variety of tissues including the brain, eye, and gonads. In an inositol phosphate assay, each receptor was functional as shown by its response to physiological doses of native GnRH peptides; two receptors showed selectivity between GnRH2 and GnRH3. Each of the four receptor genes was mapped to distinct chromosomes. Our phylogenetic and syntenic analysis segregated the four zebrafish GnRH receptors into two distinct phylogenetic groups that are separate gene lineages conserved throughout vertebrate evolution. We suggest the maintenance of four functional GnRH receptors in zebrafish compared with only one in humans may depend either on subfunctionalization or neofunctionalization in fish compared with mammalian GnRH receptors. The differences in structure, location, and response to GnRH forms strongly suggests that the four zebrafish GnRH receptors have novel functions in addition to the conventional activation of the pituitary gland in the reproductive axis.
所有脊椎动物的繁殖都需要脑激素促性腺激素释放激素（GnRH）来激活一系列导致配子发生的事件。迄今为止，所有研究过的脊椎动物在大脑的特定但不同的神经元中都有一到三种形式的 GnRH。此外，每种脊椎动物中至少有一种 GnRH 受体存在，用于激活靶细胞内的特定生理事件。人类拥有两种类型的 GnRH（GnRH1 和 GnRH2），但只有一种功能性 GnRH 受体。斑马鱼，Danio rerio，也有两种类型的 GnRH（GnRH2 和 GnRH3），尽管与人类不同，斑马鱼似乎在其基因组中有四种不同的 GnRH 受体。为了描述单一物种内多种 GnRH 受体的生物学意义，我们从斑马鱼中克隆了四种 GnRH 受体 cDNA，并比较了它们的结构、表达和细胞生理学。斑马鱼受体是 7 跨膜 G 蛋白偶联受体，四种受体之间的氨基酸序列同一性在 45%至 71%之间。与人类 GnRHR、绿猴 II 型 GnRHR 和两种金鱼 GnRHR 相比，斑马鱼 GnRHRs 的七个螺旋之间观察到高序列相似性。此外，在细胞外和细胞内结构域中存在用于假定配体结合、二硫键形成、N-糖基化和 G 蛋白偶联的关键氨基酸。四种斑马鱼受体在包括大脑、眼睛和性腺在内的各种组织中表达。在肌醇磷酸测定中，每个受体都具有功能性，因为它对生理剂量的天然 GnRH 肽有反应；两种受体显示出 GnRH2 和 GnRH3 之间的选择性。四种受体基因都被映射到不同的染色体上。我们的系统发育和基因同线性分析将四种斑马鱼 GnRH 受体分为两个不同的系统发育群，这是在脊椎动物进化过程中保守的两个独立基因谱系。我们认为，与人类相比，斑马鱼中维持四种功能性 GnRH 受体可能取决于鱼类中 GnRH 受体的亚功能化或新功能化，而不是哺乳动物 GnRH 受体。结构、位置和对 GnRH 形式的反应的差异强烈表明，除了传统激活生殖轴中的垂体外，四种斑马鱼 GnRH 受体还具有新的功能。
