Synthesis and processing in vivo of the novel mouse thyrotropin beta-presubunit that contains an NH2-terminal...

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.