SYNTHESIS AND PROCESSING IN-VIVO OF THE NOVEL MOUSE THYROTROPIN BETA-PRESUBUNIT THAT CONTAINS AN NH2-TERMINAL EXTENSION SEQUENCE

Expression of the single mouse TSHbeta gene gives rise to multiple mRN As, and we have previously shown that in vitro, one of these mRNAs giv es rise to a novel TSH beta-presubunit due to initiation of translatio n at an in-frame start site unique to this mRNA which is upstream of t he normal start site. The novel presubunit contains a 17-amino acid NH 2-terminal extension sequence compared to the normal presubunit. Altho ugh this extension sequence does not have the characteristics of a nor mal signal sequence, the novel TSH beta-presubunit was processed in vi tro by microsomal membranes. In this study we have examined the transl ation product of this mRNA in intact cells and whether in vivo it give s rise to a processed secreted TSH beta-subunit that has an NH2-termin al sequence different from that of the established TSH beta-subunit. F irstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it w as found that immunoprecipitable TSH dimer was secreted into the mediu m 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 transientl y transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when t he TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA enc oding the extended presubunit in which the down-stream AUG had been el iminated by site-specific mutagenesis. This demonstrated that the up-s tream translation start site was used in the intact cell and that secr eted TSH beta-presubunit 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 endoprote inase LysC, the NH2-terminal fragments were similar in size, suggestin g that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate tha t the longer TSH beta-presubunit is synthesized in vivo and strongly s uggest that it is processed in the intact cell to give a mature secret ed TSH beta-subunit indistinguishable from that derived from the norma l TSH beta-presubunit.