Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop
Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop
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DOI：
10.1124/mol.107.035477
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发表时间：
2007-07
影响因子：
3.6
通讯作者：
M. Ushiyama;R. Ikeda;H. Sugawara;Morikatsu Yoshida;K. Mori;K. Kangawa;Kazuhiko Inoue;Katsushi Yamada;A. Miyata
中科院分区：
文献类型：
--
作者：
M. Ushiyama;R. Ikeda;H. Sugawara;Morikatsu Yoshida;K. Mori;K. Kangawa;Kazuhiko Inoue;Katsushi Yamada;A. Miyata
腺苷酸环化酶激活多肽（PACAP）是一种多效性神经肽，具有多种生理功能。PACAP特异性受体PAC 1有几种变体，主要由编码第一胞外（EC 1）结构域和第三胞内胞质（IC 3）环的mRNA区域中的选择性剪接产生。EC 1结构域和IC 3环中可变剪接事件的组合对下游信号传导的影响尚未阐明。在这项研究中，我们已经使用半定量逆转录-聚合酶链反应（RT-PCR），以检查四个PAC 1异构体在小鼠的组织分布。然后，我们建立了细胞系组成型表达的每一个PAC 1亚型和特征的结合特性的每个亚型的PACAP-38，血管活性肠多肽（VIP），和PAC 1特异性激动剂maxadilan，以及产生的影响，两个主要的细胞内信号传导途径：cAMP的生产和细胞内钙离子浓度的变化。结果表明，IC 3环的变体影响配体对受体的结合亲和力，而EC 1结构域的变体主要影响PAC 1下游的细胞内信号传导。因此，这项研究表明，在EC 1结构域和IC 3环的选择性剪接事件的组合创建了各种各样的PAC 1亚型，这反过来可能有助于PACAP的功能多效性。本研究不仅有助于了解PACAP的多种功能，而且有助于阐明G蛋白偶联受体的结构与功能之间的关系。
Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.