Agnathan VIP, PACAP and Their Receptors: Ancestral Origins of Today's Highly Diversified Forms

VIP and PACAP are pleiotropic peptides belonging to the secretin superfamily of brain-gut peptides and interact specifically with three receptors (VPAC1, PAC1 and VPAC2) from the class II B G protein-coupled receptor family. There is immense interest regarding their molecular evolution which is often described closely alongside gene and/or genome duplications. Despite the wide array of information available in various vertebrates and one invertebrate the tunicate, their evolutionary origins remain unresolved. Through searches of genome databases and molecular cloning techniques, the first lamprey VIP/PACAP ligands and VPAC receptors are identified from the Japanese lamprey. In addition, two VPAC receptors (VPACa/b) are identified from inshore hagfish and ligands predicted for sea lamprey. Phylogenetic analyses group these molecules into their respective PHI/VIP, PRP/PACAP and VPAC receptor families and show they resemble ancestral forms. Japanese lamprey VIP/PACAP peptides synthesized were tested with the hagfish VPAC receptors. hfVPACa transduces signal via both adenylyl cylase and phospholipase C pathways, whilst hfVPACb was only able to transduce through the calcium pathway. In contrast to the widespread distribution of VIP/PACAP ligands and receptors in many species, the agnathan PACAP and VPAC receptors were found almost exclusively in the brain. In situ hybridisation further showed their abundance throughout the brain. The range of VIP/PACAP ligands and receptors found are highly useful, providing a glimpse into the evolutionary events both at the structural and functional levels. Though representative of ancestral forms, the VIP/PACAP ligands in particular have retained high sequence conservation indicating the importance of their functions even early in vertebrate evolution. During these nascent stages, only two VPAC receptors are likely responsible for eliciting functions before evolving later into specific subtypes post-Agnatha. We also propose VIP and PACAP's first functions to predominate in the brain, evolving alongside the central nervous system, subsequently establishing peripheral functions.

血管活性肠肽（Vasoactive Intestinal Peptide，VIP）与垂体腺苷酸环化酶激活肽（Pituitary Adenylate Cyclase-Activating Polypeptide，PACAP）属于脑肠肽促胰液素超家族，是一类多效性肽类物质，可与B类II型G蛋白偶联受体家族的3种受体（VPAC1、PAC1及VPAC2）特异性结合。学界对其分子进化机制抱有极高研究热情，该进化过程通常与基因及（或）基因组重复事件紧密关联。尽管目前已在多种脊椎动物以及1种无脊椎动物——被囊动物中积累了海量研究数据，但二者的进化起源仍未得到明确阐释。本研究通过基因组数据库检索与分子克隆技术，首次从日本七鳃鳗中鉴定出VIP/PACAP配体与VPAC受体；此外，还从近岸盲鳗中鉴定出2种VPAC受体（VPACa/b），并预测了海七鳃鳗的配体序列。系统发育分析结果显示，这些分子可分别归入PHI/VIP、PRP/PACAP及VPAC受体家族，且其结构特征与祖先型分子高度相似。研究人员合成了日本七鳃鳗的VIP/PACAP肽类，并对其与盲鳗VPAC受体的信号转导活性进行了检测：hfVPACa可通过腺苷酸环化酶与磷脂酶C双通路完成信号转导，而hfVPACb仅能通过钙信号通路实现信号转导。与VIP/PACAP配体及受体在诸多物种中广泛分布的特征不同，无颌类脊椎动物的PACAP与VPAC受体几乎仅在脑组织中表达；原位杂交实验进一步证实，二者在全脑范围内均有丰富分布。本次鉴定得到的一系列VIP/PACAP配体与受体具有极高研究价值，为我们从结构与功能层面窥探进化事件提供了重要视角。尽管属于祖先型分子，尤其是VIP/PACAP配体仍保持了较高的序列保守性，这表明其功能在脊椎动物进化早期便已具备核心意义。在脊椎动物进化的早期阶段，仅存在2种VPAC受体负责介导相关生理功能，后续才在无颌类之后进化出多种特异性亚型。我们还提出，VIP与PACAP的原始功能主要在脑部发挥，并随中枢神经系统一同演化，随后才逐步建立起外周组织中的生理功能。