Novel splice variants, of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance
Novel splice variants, of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance
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DOI：
10.1210/en.143.7.2680
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发表时间：
2002-07-01
期刊：
影响因子：
4.8
通讯作者：
Anouar, Y
中科院分区：
文献类型：
--
作者：
Alexandre, D;Vaudry, H;Anouar, Y
垂体腺苷环化酶激活多肽(PACAP)通过激活两种G蛋白偶联受体发挥不同的作用，一种是与PACAP有高亲和力的受体PAC1-R，另两种是与PACAP和血管活性肠肽有类似亲和力的受体VPAC1-R和VPAC2-R。在这里，我们报告了蛙类Rana ridibunda中PAC1-R和新的剪接变异体的特征。青蛙PAC1-R与人类PAC1-R有78%的同源性，在中枢神经系统中高度表达。青蛙受体的两个剪接变体在第三个细胞内环中显示额外的氨基酸盒被鉴定。PAC1-R25携带一个25个氨基酸的插入，与哺乳动物受体的HOP盒匹配，而PAC1-R41携带的盒与任何哺乳动物PAC1-R变体没有同源性。PAC1-R的第三个剪接变异体也被鉴定出来，它显示了一个完全不同的细胞内C-末端结构域，称为PAC1-RMC。细胞内cAMP形成的测定表明，PACAP对PAC1-R、PAC1-R25和PAC1-R41具有相似的激活作用。相反，PACAP不能刺激转导PAC1 RMC的细胞的腺苷环化酶。PAC1-R或PAC1-RMC与绿色荧光蛋白的融合表明，这两种受体在转基因细胞中都有表达并定位于质膜。不同的PAC1-R变异体在蛙脑和脊髓中高度表达，在外周组织中表达较少，只有某些亚型才能检测到。目前的数据表明，在蛙类中，PACAP可能通过不同的PAC1-R剪接变体起作用，这些剪接变体在不同组织中的G(S)蛋白偶联和丰度不同。
Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of cAMP formation in cells transfected with the cloned receptors showed that PACAP activated PAC1-R, PAC1-R25, and PAC1-R41 with similar potency. In contrast, PACAP failed to stimulate adenylate cyclase in cells transfected with PAC1 Rmc. Fusion of PAC1-R or PAC1-Rmc with the green fluorescent protein revealed that both receptors are expressed and targeted to the plasma membrane in transfected cells. The different PAC1-R variants are highly expressed in the frog brain and spinal cord and to a lesser extent in peripheral tissues, where only certain isoforms could be detected. The present data indicate that in frog, PACAP may act through different PAC1-R splice variants that differ in their G(s) protein coupling and their abundance in various tissues.