Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.
Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.
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DOI：
10.1186/1471-2148-7-221
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发表时间：
2007-11-12
影响因子：
3.4
通讯作者：
Power DM
中科院分区：
文献类型：
--
作者：
Cardoso JC;de Vet EC;Louro B;Elgar G;Clark MS;Power DM
重复基因在脊椎动物基因组中很常见。它们的持久性被认为是获得新功能（新功能化）或祖先分子功能分区（亚功能化）的结果。令人惊讶的是，很少有研究评估这种修饰的程度，尽管迄今为止在脊椎动物基因组中鉴定出了许多重复的受体和配体基因。为了研究功能在维持重复基因中的重要性，研究了真鲷（Sparus auratus）PAC 1受体，其是哺乳动物特异性受体PACAP（PADAP环化酶激活多肽）的序列同源物。这些受体属于家族2 GPCR，并且它们的大多数成员在硬骨鱼中是重复的，尽管两者在基因组中持续存在的原因尚不清楚。重复的海鲷PACAP受体基因（sbPAC 1A和sbPAC 1B），家庭2 GPCR的成员，分离和共享77%的氨基酸序列同一性。每个基因的特异性引物的RT-PCR显示，它们具有与单一哺乳动物受体的分布重叠的差异组织分布。此外，与哺乳动物一样，硬骨鱼基因经历选择性剪接，并且已经表征了PAC 1Ahop 1同种型。在其他硬骨鱼基因组中也发现了重复的正向受体，其分布特征表明其功能可能具有物种特异性。对Cos 7细胞中的parasites bPAC 1的功能分析显示，它们在哺乳动物PACAP 27和PACAP 38的存在下受到强烈刺激，而VIP（血管活性肠肽）的刺激要少得多。sbPAC 1受体同样受到刺激，（最大cAMP产生的LOGEC 50值）（sbPAC 1A和sbPAC 1B分别为-8.74 ± 0.29 M和-9.15 ± 0.21 M，P > 0.05）和PACAP 38（sbPAC 1A和sbPAC 1B分别为-8.54 ± 0.18 M和-8.92 ± 0.24 M，P > 0.05）。发现人VIP刺激sbPAC 1A（-7.23 ± 0.20 M）比sbPAC 1B（-6.57 ± 0.14 M，P < 0.05）更强，并且迄今为止尚未在鱼类基因组中鉴定的人促胰液素（SCT）对两种受体的刺激可忽略不计。功能上不同的复制sbPAC 1受体的存在与先前提出的关于复制基因的起源和维持的理论是一致的。海鲷PAC 1复制受体类似于典型的哺乳动物PAC 1，并且发现PACAP肽在刺激cAMP产生方面比VIP更有效，尽管sbPAC 1A比sbPAC 1B对VIP更敏感。这些结果与组织分布的高度分歧的模式表明，涉及neofunctionalisation的过程发生后，受体复制内的鱼类谱系，并可能占其在基因组中的持久性。进一步重复的受体及其配体的特性应该提供洞察与脊椎动物辐射相关的新的蛋白质-蛋白质相互作用的进化和功能。
Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74 ± 0.29 M and -9.15 ± 0.21 M, respectively for sbPAC1A and sbPAC1B, P > 0.05) and PACAP38 (-8.54 ± 0.18 M and -8.92 ± 0.24 M, respectively for sbPAC1A and sbPAC1B, P > 0.05). Human VIP was found to stimulate sbPAC1A (-7.23 ± 0.20 M) more strongly than sbPAC1B (-6.57 ± 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation.
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