Identification of PAC1 receptor isoform mRNAs by real-time PCR in rat suprachiasmatic nucleus
Identification of PAC1 receptor isoform mRNAs by real-time PCR in rat suprachiasmatic nucleus
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DOI：
10.1016/s0169-328x(02)00387-x
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发表时间：
2002-09-30
期刊：
影响因子：
--
通讯作者：
Sugden, D
中科院分区：
文献类型：
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作者：
Ajpru, S;McArthur, AJ;Sugden, D
垂体腺苷酸环化酶激活多肽(PACAP)与视交叉上核(SCN)内啮齿动物昼夜节律的光重置有关。PACAP可通过VPAC1、VPAC2和PAC1G蛋白偶联受体发挥作用。大鼠SCN表达PAC1和VPAC2，但不表达VPAC1。各种PAC1受体剪接变异体在配体结合亲和力和选择性、G蛋白偶联和激活信号转导途径的能力方面表现出差异。本实验采用聚合酶链式反应和同工异构体特异的方法来确定哪些PAC1变异体在大鼠SCN中表达。检测到PACI(空)异构体和一个在第三胞内环(IC3)插入一个28个氨基酸的变异体(HopI1/2)。在任何时间获得的SCN中均未检测到其他IC3变异体(HIP、HIP-HOP)、N端变异体(PAC1(Short)、PAC1(Very Short)和PAC1(3a))或跨膜II和IV不同的变异体(PAC1TM4)。建立了一种实时荧光定量聚合酶链式反应(RT-PCR)方法，检测12：12-h光：暗(L：D)周期中PAC1(空/HOP)变异体在大鼠SCN中的联合表达。PAC1mRNA的表达(PAC1(空)+PAC1(HOP))随时间变化不明显。在一天中的不同时间，这两种变异的比例也没有显著差异。这些结果表明，PACAP对SCN放电和昼夜节律行为的作用不是通过PAC1受体mRNA表达水平的变化来调节的，也不是通过具有改变的配体结合、G蛋白偶联或信号特征的PAC1受体变体的相依赖表达来调节的。(C)2002 Elsevier Science B.V.保留所有权利。
The pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the photic resetting of the rodent circadian clock in the suprachiasmatic nucleus (SCN). PACAP can exert its effects via VPAC1, VPAC2 and PAC1 G-protein coupled receptors. PAC1 and VPAC2, but not VPAC1, mRNA is expressed in rat SCN. A variety of PAC1 receptor splice variants have been described showing differences in ligand binding affinity and selectivity, G-protein coupling and ability to activate signal transduction pathways. The present experiments used PCR with isoform specific primers to determine which PAC1 variants are expressed in rat SCN. The PACI(null) isoform and a variant containing a single 28-amino acid insert in the third intracellular (IC3) loop (hopI1/2) were detected. No other IC3 variants (hip, hip-hop), N-terminal variants (PAC1(short), PAC1(very short) and PAC1(3a)) or the variant differing in transmembrane II and IV (PAC1TM4) were detected in SCN obtained at any time of day. A quantitative real-time PCR assay was established which measured combined expression of the PAC1(null/hop) variants in rat SCN during a 12:12-h light:dark (L:D) cycle. There was no significant variation of PAC1 mRNA expression (PAC1(null)+PAC1(hop)) With time of day. Nor was there a significant difference in the proportion of these two variants with time of day. These results indicate that the phase-dependency of the actions of PACAP on SCN firing and circadian behaviour are not mediated by changes in the level of expression of PAC1 receptor mRNA, nor by phase-dependent expression of PAC1 receptor variants with altered ligand binding, G-protein coupling or signalling characteristics. (C) 2002 Elsevier Science B.V. All rights reserved.