Lutz E M, Ronaldson E, Shaw P, Johnson M S, Holland P J, Mitchell R
Molecular Signalling Group, Department of Bioscience, University of Strathclyde, Royal College, 204 George St., Glasgow G1 1XW, UK.
Mol Cell Neurosci. 2006 Feb;31(2):193-209. doi: 10.1016/j.mcn.2005.09.008. Epub 2005 Oct 14.
Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.
采用逆转录聚合酶链反应（RT-PCR）和定量PCR技术，在六种神经母细胞瘤细胞系以及人类胚胎和成人脑组织中测定了血管活性肠肽（VIP）受体（VPAC）和垂体腺苷酸环化酶激活肽（PACAP）1型受体亚型的表达。发现氨基末端结构域缺失21个氨基酸序列的PAC1受体剪接变体是神经母细胞瘤细胞系中的主要受体变体，并且与成人大脑相比，在胚胎大脑中也高度表达。在四种神经母细胞瘤细胞系中，VIP和PACAP以不同的效力和最大刺激水平刺激环磷酸腺苷（cAMP）的产生。在SH-SY5Y细胞中记录到每种肽对cAMP的高效力和最大刺激，表明存在高亲和力的VIP和PACAP受体。在SH-SY5Y细胞系中对特定的VPAC和PAC1受体亚型进行了进一步表征，在该细胞系中，除了已知的PAC1受体剪接变体和VPAC2受体外，还鉴定出了许多新的PAC1受体剪接变体。VPAC2受体的相对低水平表达以及SH-SY5Y细胞对VPAC2受体特异性激动剂Ro 25-1553的反应性较差，表明该受体对观察到的VIP反应没有显著贡献。当在COS 7细胞中表达单个PAC1受体亚型时，与具有完整结构域的受体相比，在大多数缺乏21个氨基酸氨基末端结构域序列的PAC1受体变体中，VIP激活cAMP产生的能力增加了50倍以上。在PACAP-38的效力方面观察到较小的变化。在肌醇磷酸反应中也观察到类似趋势，在每种情况下激动剂效力均低于cAMP产生的效力。本研究结果表明，PAC1受体中不同的氨基末端和细胞内环3剪接变体的组合决定了激动剂（特别是VIP）激活信号通路的能力。VIP在大多数具有短氨基末端结构域的PAC1受体上具有相当大的效力，因此这些受体可能介导VIP和PACAP的生理效应。此外，在胚胎和成熟神经系统之间，不同PAC1受体氨基末端剪接变体的表达可能存在表型转换，这表明对该事件的调节可能在VIP/PACAP功能中起重要作用，特别是在发育中的神经系统中。
