Pisegna J R, Moody T W, Wank S A
Digestive Diseases Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ann N Y Acad Sci. 1996 Dec 26;805:54-64; discussion 64-6. doi: 10.1111/j.1749-6632.1996.tb17473.x.
Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the VIP/secretin/glucagon family, is present in the hypothalamus, anterior pituitary, and adrenal gland where it regulates hormone release, in the GI tract where it modulates motility, and in human tumoral cell lines where it shows a growth-promoting effect. It is now appreciated that alternative splicing of two exons of the rat PACAP-R gene generate four major rPACAP-R splice variants that are differentially expressed in tissues and variably coupled to intracellular second messengers. Because of the potential implications of these findings in human physiology, we cloned the hPACAP-R gene. Similar to the rat, two exons (SV-1 and SV-2) are alternatively spliced to account for four major hPACAP-R receptor splice variants. These splice variants (hPACAP-R-null, hPACAP-R-SV1, hPACAP-R-SV2, hPACAP-R-SV-3) were cloned from a human frontal cortex cDNA library, stably transfected in NIH/ 3T3 cells and each characterized for ligand affinity, stimulation of adenylate cyclase (AC) and phospholipase C (PLC), and ligand-induced expression of the proto-oncogenes, c-fos, and c-myc. Stably transfected NIH/3T3 cells expressing similar numbers of receptors of the four splice variants showed nearly identical responses for ligand affinity and potency for P-38- and P-27-stimulated increases in cAMP and total inositol phosphates. However, each receptor splice variant differed in their ligand-stimulated efficacy for total inositol phosphate stimulation. The hPACAP-R-SV2 showed the greatest efficacy for stimulating phospholipase C that was approximately seven-fold greater than the hPACAP-R-SV1, twofold greater than the hPACAP-R-Null, and 1.5-fold greater than the hPACAP-R-SV-3 splice variants. To determine whether the splice variants also differ in their ability to stimulate immediate early gene expression, c-fos and c-myc transcripts were assayed by Northern blot and quantified by densitometry. PACAP-38 increased c-fos and c-myc expression for all four of the receptor splice variants that paralleled the efficacy for PLC stimulation, with the the SV-2 splice variant showing the greatest stimulation. These results show that the hPACAP-R-SV2 exhibits enhanced efficacy for coupling to both PLC and activation of the protooncogenes, c-fos and c-myc suggesting a novel and potentially important mechanism for differentially activating signal transduction pathways that influence cellular growth and differentiation.
垂体腺苷酸环化酶激活多肽（PACAP）是一种属于血管活性肠肽/促胰液素/胰高血糖素家族的神经肽，存在于下丘脑、垂体前叶和肾上腺，在这些部位它调节激素释放；存在于胃肠道，在胃肠道调节蠕动；还存在于人类肿瘤细胞系，在肿瘤细胞系中它具有促生长作用。现在人们认识到，大鼠PACAP-R基因的两个外显子的可变剪接产生了四种主要的rPACAP-R剪接变体，它们在组织中差异表达，并与细胞内第二信使有不同的偶联。由于这些发现对人类生理学可能有影响，我们克隆了hPACAP-R基因。与大鼠相似，两个外显子（SV-1和SV-2）进行可变剪接，产生四种主要的hPACAP-R受体剪接变体。这些剪接变体（hPACAP-R-null、hPACAP-R-SV1、hPACAP-R-SV2、hPACAP-R-SV-3）从人额叶皮质cDNA文库中克隆出来，稳定转染到NIH/3T3细胞中，并对每种变体的配体亲和力、腺苷酸环化酶（AC）和磷脂酶C（PLC）的刺激以及原癌基因c-fos和c-myc的配体诱导表达进行了表征。稳定转染表达相似数量的四种剪接变体受体的NIH/3T3细胞，对配体亲和力以及P-38和P-27刺激的cAMP增加和总肌醇磷酸的效力显示出几乎相同的反应。然而，每种受体剪接变体在其配体刺激的总肌醇磷酸刺激效力方面有所不同。hPACAP-R-SV2在刺激磷脂酶C方面显示出最大的效力，比hPACAP-R-SV1大约高7倍，比hPACAP-R-Null高2倍，比hPACAP-R-SV-3剪接变体高1.5倍。为了确定剪接变体在刺激即刻早期基因表达的能力上是否也存在差异，通过Northern印迹法检测c-fos和c-myc转录本，并通过光密度测定法定量。PACAP-38增加了所有四种受体剪接变体的c-fos和c-myc表达，这与PLC刺激的效力平行，其中SV-2剪接变体显示出最大的刺激。这些结果表明，hPACAP-R-SV2在与PLC偶联以及激活原癌基因c-fos和c-myc方面表现出增强的效力，这表明存在一种新的且可能重要的机制，用于差异激活影响细胞生长和分化的信号转导途径。
