Schneider M J, Davey J C, Galton V A
Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001.
Endocrinology. 1993 Dec;133(6):2488-95. doi: 10.1210/endo.133.6.8243269.
Thyroid hormone (TH) receptor number in red blood cells (RBCs) from Rana catesbeiana (RC) tadpoles increases 4-fold during both spontaneous and TH-induced metamorphosis, an effect that we have previously shown to be preceded by an increase in the level of c-erbA-related mRNA. The goals of the present study were to obtain an RC c-erbA alpha cDNA that contains the entire open reading frame for a putative TH receptor protein, to determine if this protein has characteristics typical of a TH receptor, and to assess its contribution to the developmentally related increase in TH receptor number. To accomplish this, the missing 5'-sequence of a previously isolated partial RC c-erbA alpha cDNA (RC12) was synthesized by polymerase chain reaction (PCR) and spliced to RC12 to yield a 1490-basepair cDNA (RC15) that contained the entire coding sequence of the receptor protein. Transcription of RC15 followed by translation of its mRNA in a rabbit reticulolysate system yielded a 50-kilodalton protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds T3 with high affinity (Kd, approximately 0.1 nM), and its affinity for T3 is at least 5 times that for T4. The results of cotransfection studies indicate that RC15 can function as a TH receptor; when COS cells were cotransfected with a construct consisting of RC15 cloned in the expression vector CMV4 and TK28 mult, a construct containing rat GH gene TH response element sequences up-stream of a chloramphenicol acetyltransferase reporter gene, chloramphenicol acetyltransferase activity is expressed in the presence, but not in the absence, of T3. To determine whether RBCs contain any c-erbA beta mRNA transcripts that might contribute to the developmentally related increase in the transcripts detected using RC c-erbA alpha cDNAs, alpha- and beta-specific cDNAs were synthesized by PCR and used as probes in a variety of hybridization assays. In all experiments using conditions in which c-erbA beta transcripts were detectable in other tissues, there was no evidence that tadpole RBCs contained such species. Lack of any beta-specific transcripts was confirmed by PCR, using as template cDNA prepared by reverse transcription of RC RBC RNA. It was also noted that the RBC at metamorphic climax is the tissue with the highest content of alpha-specific c-erbA transcripts. It is concluded that the c-erbA alpha gene encodes a TH receptor, and that only the alpha-gene is expressed in tadpole RBCs and subject to regulation during development and by TH.
牛蛙蝌蚪红细胞中的甲状腺激素（TH）受体数量在自发变态和TH诱导的变态过程中增加了4倍，我们之前已经表明，这一效应之前伴随着c-erbA相关mRNA水平的升高。本研究的目的是获得一个牛蛙c-erbAα cDNA，其包含假定的TH受体蛋白的完整开放阅读框，确定该蛋白是否具有TH受体的典型特征，并评估其对TH受体数量与发育相关增加的贡献。为了实现这一目标，通过聚合酶链反应（PCR）合成了先前分离的部分牛蛙c-erbAα cDNA（RC12）缺失的5'序列，并将其与RC12拼接，得到一个1490碱基对的cDNA（RC15），其包含受体蛋白的完整编码序列。RC15转录后，其mRNA在兔网织红细胞裂解液系统中翻译，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上产生一个50千道尔顿的蛋白质。该蛋白以高亲和力（Kd，约0.1 nM）结合T3，其对T3的亲和力至少是对T4的5倍。共转染研究结果表明，RC15可作为TH受体发挥作用；当COS细胞与由克隆在表达载体CMV4中的RC15和TK28 mult组成的构建体共转染时，后者是一个在氯霉素乙酰转移酶报告基因上游包含大鼠GH基因TH反应元件序列的构建体，在有T3存在但无T3时不存在的情况下，氯霉素乙酰转移酶活性得以表达。为了确定红细胞是否含有任何可能导致使用牛蛙c-erbAα cDNA检测到的转录本与发育相关增加的c-erbAβ mRNA转录本，通过PCR合成了α和β特异性cDNA，并将其用作各种杂交试验的探针。在所有使用在其他组织中可检测到c-erbAβ转录本的条件的实验中，没有证据表明蝌蚪红细胞含有此类转录本。通过PCR，以牛蛙红细胞RNA逆转录制备的cDNA为模板，证实了不存在任何β特异性转录本。还注意到，变态高峰期的红细胞是α特异性c-erbA转录本含量最高的组织。结论是，c-erbAα基因编码一种TH受体，并且只有α基因在蝌蚪红细胞中表达，并在发育过程中以及受TH调控。
