Too C K, Vickaryous N, Boudreau R T, Sangster S M
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Endocrinology. 2001 Mar;142(3):1357-67. doi: 10.1210/endo.142.3.8041.
A full-length, PRL-inducible complementary DNA (cDNA) encoding a novel, nuclear-targeted carboxypeptidase D isoform (designated CPD-N) was identified in the rat PRL-dependent Nb2-11C and PRL-independent Nb2-Sp lymphoma cell lines by differential display. The CPD-N cDNA (3751 bp) has 99% (3582/3583) homology with rat carboxypeptidase D (CPD; 4377 bp). In comparison to the rat CPD cDNA (ORF of 4134 bp; 180-kDa protein), CPD-N was shorter by approximately 600 bases but contained 148 unique bases at the 5'-end to give an ORF of 3399 bp. RT-PCR with primers specific to the 5'-end of CPD-N or to CPD showed that the CPD-N transcript was expressed in the Nb2-11C and Nb2-Sp cells but was not detected in rat brain or lung. Conversely, the CPD transcript was expressed in rat brain but was not detected in the two Nb2 cell lines. CPD-N expression (7.5-kb messenger RNA) was stimulated by PRL (10 ng/ml) and/or by interleukin-2 (24 U/ml) in Nb2-11C and Nb2-Sp cells. Most rat tissues expressed multiple CPD transcripts (7.5, 4.1, and 2 kb). Curiously, CPD transcripts were low or undetectable in male rat liver but readily detected in female liver, suggesting that sex-specific hormone levels may regulate its expression. Indeed, CPD expression in the PRL-responsive HepG2 hepatoma and MCF-7 breast cancer cell lines was low in control cells but was markedly stimulated by PRL after 3 h. Consistent with the shorter ORF of CPD-N, Western analysis detected proteins of smaller molecular sizes of 160 kDa (abundant) and 117 kDa (weak) in the Nb2-11C cells. The Nb2-Sp cells expressed a single and abundant 117-kDa protein, implicating differential protein processing in the two cell lines. Rat CPD has been reported to colocalize with the trans-Golgi network marker TGN38. Subcellular fractionation showed predominant nuclear localization of CPD-N and trace amounts were detected in the 100,000 x g microsomal fraction after PRL treatment (4 h); in contrast, TGN38 was found only in the microsomal fraction at this time. In cells treated with PRL for 24 h, immunofluorescent confocal microscopy showed nuclear and cytoplasmic distribution of CPD-N. Cytoplasmic CPD-N colocalized with TGN-38 whereas nuclear CPD-N had a mesh-like distribution and colocalized with nuclear lamin B.
通过差异显示技术，在大鼠泌乳素依赖的Nb2-11C和泌乳素非依赖的Nb2-Sp淋巴瘤细胞系中，鉴定出一种全长的、泌乳素诱导的互补DNA（cDNA），它编码一种新的、定位于细胞核的羧肽酶D同工型（命名为CPD-N）。CPD-N cDNA（3751 bp）与大鼠羧肽酶D（CPD；4377 bp）具有99%（3582/3583）的同源性。与大鼠CPD cDNA（开放阅读框为4134 bp；180 kDa蛋白）相比，CPD-N短约600个碱基，但在5'端含有148个独特碱基，开放阅读框为3399 bp。用特异于CPD-N 5'端或CPD的引物进行逆转录聚合酶链反应（RT-PCR）表明，CPD-N转录本在Nb2-11C和Nb2-Sp细胞中表达，但在大鼠脑或肺中未检测到。相反，CPD转录本在大鼠脑中表达，但在两种Nb2细胞系中未检测到。在Nb2-11C和Nb2-Sp细胞中，泌乳素（10 ng/ml）和/或白细胞介素-2（24 U/ml）可刺激CPD-N表达（7.5 kb信使RNA）。大多数大鼠组织表达多种CPD转录本（7.5、4.1和2 kb）。奇怪的是，雄性大鼠肝脏中的CPD转录本水平较低或无法检测到，但在雌性肝脏中很容易检测到，这表明性别特异性激素水平可能调节其表达。事实上，在泌乳素反应性的HepG2肝癌细胞系和MCF-7乳腺癌细胞系中，对照细胞中CPD表达较低，但3小时后泌乳素可显著刺激其表达。与CPD-N较短的开放阅读框一致，蛋白质印迹分析在Nb2-11C细胞中检测到分子量较小的蛋白质，分别为160 kDa（丰富）和117 kDa（较弱）。Nb2-Sp细胞表达一种单一且丰富的117 kDa蛋白，这表明两种细胞系中存在不同的蛋白质加工过程。据报道，大鼠CPD与反式高尔基体网络标志物TGN38共定位。亚细胞分级分离显示，CPD-N主要定位于细胞核，泌乳素处理（4小时）后，在100,000×g微粒体组分中检测到微量；相比之下，此时TGN38仅在微粒体组分中发现。在泌乳素处理24小时的细胞中，免疫荧光共聚焦显微镜显示CPD-N在细胞核和细胞质中均有分布。细胞质中的CPD-N与TGN-38共定位，而细胞核中的CPD-N呈网状分布，并与核纤层蛋白B共定位。
