Parathyroid hormone-related protein: evidence for isoform- and tissue-specific posttranslational processing.
Parathyroid hormone-related protein: evidence for isoform- and tissue-specific posttranslational processing.
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甲状旁腺激素相关蛋白：异构体和组织特异性翻译后加工的证据。
DOI：
10.1021/bi00189a054
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Broadus,AE
中科院分区：
文献类型：
--
作者：
Yang,KH;dePapp,AE;Soifer,NE;Dreyer,BE;Wu,TL;Porter,SE;Bellantoni,M;Burtis,WJ;Insogna,KL;Broadus,AE
甲状旁腺激素相关蛋白（PTHrP）在恶性肿瘤中表达，并导致恶性肿瘤的体液性高钙血症综合征。它也在多种非恶性组织中表达，在这些组织中似乎起着不同的旁分泌和/或自分泌作用。人PTHrP基因编码三种cDNA预测的139、141和173个氨基酸的初始翻译产物。大多数人类细胞系含有编码所有三种PTHrP亚型的mRNA。这三种高度相似的转录本存在的生理学原理尚不清楚。为了确定来源于这三种转录物的蛋白产物是否不同，我们用编码人PTHrP（1 -139）、PTHrP（1 -141）和PTHrP（1 -173）的cDNA分别转染中国仓鼠卵巢（CHO）细胞和大鼠胰岛素瘤（RIN）细胞。然后用反相HPLC对细胞提取物和条件培养基进行色谱分析，并用区域特异性PTHrP免疫测定法进行分析。正如我们先前在SKRC-1（肾细胞癌）和RIN（1-141）细胞中观察到的那样，在所有六种细胞系中鉴定出多个氨基末端PTHrP种类以及单独的中间区域PTHrP种类。此外，用PTHrP（1 -139）构建体转染的CHO和RIN细胞系都含有以前未识别的羧基末端片段，其与PTHrP（109-138）抗血清反应。该羧基末端片段在物理上不同于先前发现的中间区片段，并且也存在于条件培养基中，表明其是分泌形式，而不是生物合成中间体或降解产物。令人惊讶的是，用PTHrP（1 -141）或PTHrP（1-173）转染的RIN和CHO细胞几乎不含这种羧基末端片段，这表明PTHrP存在同种型特异性蛋白质加工。此外，虽然RIN细胞产生一个主要的氨基末端物种，CHO细胞含有两个，约等摩尔，氨基末端物种，表明存在细胞特异性蛋白质加工。这些研究表明PTHrP的翻译后加工是高度复杂的。具体地说，（a）含有三种PTHrP转录物中的每一种的细胞系产生多个氨基末端PTHrP分泌形式，以及中间区形式;（B）产生中间区片段的Arg 37切割发生在高尔基体中，因为组成型和调节型分泌细胞类型都能够进行这种切割;（c）分泌以前未识别的PTHrP羧基末端片段;和（d）PTHrP的加工似乎是同种型和细胞特异性的。这些片段中每个片段的完整结构测定对于了解甲状旁腺激素相关蛋白（PTHrP）生理学和病理生理学至关重要。甲状旁腺激素相关蛋白（PTHrP）最初从与恶性体液高钙血症综合征（HHM）相关的人类癌症中分离出来（Bilezikian，1990; Broadus & Stewart，1994; Halloran & Nissenson，1992; Stewart & Broadus，1990 a，B; Strewler & Nissenson，1990）。从那时起，PTHrP蛋白、mRNA或两者的存在已经在许多正常的、非恶性的人和动物组织中被证明（Bilez-2005）。
Revised Manuscript Received April 5, 1994• abstract: Parathyroid hormone-related protein (PTHrP) is expressed by malignant tumors and leads to the syndrome of humoral hypercalcemia of malignancy. It is also expressed by a wide varietyof nonmalignant tissues, in which it appears to playdistinct paracrine and/or autocrine roles. The human PTHrP gene encodes three cDNA-predicted initial translational products of 139, 141, and 173 amino acids. Most human cell lines contain mRNAs encoding all three PTHrP isoforms. The physiological rationalefor the existence of these three highly similar transcripts is unknown. In order to determine whether the protein products derived from these three transcripts differ, we transfected Chinese hamster ovary (CHO) cells and rat insulinoma (RIN) cells individually with cDNAs encoding human PTHrP (l-139), PTHrP (l-141), and PTHrP (l-173). Cell extractsand conditionedmedium were then chromatographed using reversed-phase HPLC and analyzed using region-specific PTHrP immunoassays. As we had previously observed in SKRC-1 (renal cell carcinoma) and RIN (1-141) cells, multiple amino-terminal PTHrP species as well as a separate midregion PTHrP species were identified in all six celllines. In addition, both CHO and RIN cell lines transfected with the PTHrP (l-139) construct contained a previously unrecognized carboxy-terminal fragment that reacted with a PTHrP (109-138) antiserum. This carboxy-terminal fragment was physically distinct from the midregion fragment discovered earlier and was also present in conditioned medium, indicating that it is a secretory form, rather than a biosynthetic intermediate or a degradation product. Surprisingly, RIN and CHO cells transfected with PTHrP (l-141) or-(1-173) contained little of this carboxy-terminal fragment, suggesting that isoform-specific protein processing exists for PTHrP. In addition, while RIN cells produced a single predominant amino-terminal species, CHO cells contained two, approximately equimolar, amino-terminal species, indicating the existence of cell-specific protein processing. These studies indicate that the posttranslational processing of PTHrP is highly complex. Specifically,(a) multiple amino-terminal PTHrP secretory forms, as well as a midregion form, are generated by cell lines containing each of the three PTHrP transcripts;(b) the Arg37 cleavage that generates the midregion fragment occurs in the Golgi apparatus, as both constitutive and regulated secretory cell types are capable of performing this cleavage;(c) a previously unrecognized carboxy-terminal fragment of PTHrP is secreted; and (d) processing of PTHrP appears to be both isoform-and cell-specific. Complete structural determination of each of these fragments is critical to understanding PTHrP physiology and pathophysiology.Parathyroid hormone-related protein (PTHrP) was orig-inally isolated from human cancers associated with the syndrome of humoral hypercalcemia of malignancy (HHM)(Bilezikian, 1990; Broadus & Stewart, 1994; Halloran & Nissenson, 1992; Stewart & Broadus, 1990a, b; Strewler & Nissenson, 1990). Since then, the presence of either PTHrP protein, mRNA, or both has been documented in a multitude of normal, non-malignant human and animal tissues (Bilez-