Januszeski M M, Gabriel J L, Shennan K I, Docherty K, Gurr J A
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Endocrinology. 1994 Apr;134(4):1859-67. doi: 10.1210/endo.134.4.8137753.
Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.
单一小鼠促甲状腺激素β基因的表达可产生多种mRNA，我们之前已经表明，在体外，这些mRNA中的一种可产生一种新型促甲状腺激素β前体亚基，这是由于在该mRNA特有的框内起始位点（位于正常起始位点上游）起始翻译所致。与正常前体亚基相比，这种新型前体亚基含有一个17个氨基酸的NH2末端延伸序列。尽管这个延伸序列不具备正常信号序列的特征，但这种新型促甲状腺激素β前体亚基在体外可被微粒体膜加工处理。在本研究中，我们检测了这种mRNA在完整细胞中的翻译产物，以及在体内它是否会产生一种经加工分泌的促甲状腺激素β亚基，其NH2末端序列与已确定的促甲状腺激素β亚基不同。首先，将编码α前体亚基以及正常或新型促甲状腺激素β前体亚基的mRNA显微注射到非洲爪蟾卵母细胞中，结果发现，无论用于促甲状腺激素β亚基合成的是哪种mRNA，均可分泌出可免疫沉淀的促甲状腺激素二聚体至培养基中。然而，当促甲状腺激素β亚基源自延长型促甲状腺激素β前体亚基时，获得的促甲状腺激素较少。其次，当用表达α前体亚基以及正常或新型促甲状腺激素β前体亚基的质粒瞬时转染COS细胞时，无论促甲状腺激素β亚基源自哪种前体亚基，均可获得分泌型促甲状腺激素。当促甲状腺激素β前体亚基源自编码延长型前体亚基的mRNA（其中下游AUG已通过定点诱变被消除）时，也可获得促甲状腺激素二聚体。这表明上游翻译起始位点在完整细胞中被使用，且分泌型促甲状腺激素β亚基源自延长型前体亚基，而非由翻译通读至下游AUG产生的正常前体亚基。当用内肽酶LysC消化源自正常和延长型促甲状腺激素β前体亚基的分泌型促甲状腺激素β亚基时，NH2末端片段大小相似，这表明NH2末端延伸对信号肽酶的切割位点影响很小（如果有影响的话）。因此，我们的数据表明，较长的促甲状腺激素β前体亚基在体内合成，并且有力地表明它在完整细胞中被加工处理，以产生一种成熟的分泌型促甲状腺激素β亚基，与源自正常促甲状腺激素β前体亚基的促甲状腺激素β亚基无法区分。
