BOTH THE 5'-NONCODING AND 3'-NONCODING REGIONS OF THE THYROTROPIN RECEPTOR MESSENGER-RIBONUCLEIC-ACID INFLUENCE THE LEVEL OF RECEPTOR PROTEIN EXPRESSION IN TRANSFECTED MAMMALIAN-CELLS

The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investig ated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by de leting either the 5' untranslated region (UTR), the 3'UTR, or both UTR s. The 5'UTR contains two false AUG initiation codons followed by a st op codon. The cDNAs in the eukaryotic expression vector pSV2-NEO-ECE, as well as the 5'3'UTR-truncated cDNA in pSVL, were stably transfected into Chinese hamster ovary cells. Pools of more than 100 colonies wer e studied in order to minimize insertion site-dependent variation in t he level of expression. Scatchard analysis of TSH binding indicated th at the number of receptors on the surface of Chinese hamster ovary cel ls expressing the wild-type transcript (similar to 16,000/cell) increa sed approximately a-fold with 5'UTR deletion, approximately 5-fold wit h 3'UTR deletion, and approximately 10-fold with both 5'UTR and 3'UTR deletion. TSH binding affinities of all constructs were in the range o f 2-5 x 10(-10) M. No significant difference was evident between the 5 '3'UTR truncated cDNAs in the two different vectors, pSV2-NEO-ECE and pSVL. The increase in the amplitude of the cAMP response to TSH stimul ation was commensurate with the number of receptors expressed on the s urface of the different cell lines. Truncation of the 5'UTR did not al ter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as we ll as the 5'3'UTR-deleted transcript, increased approximately 4-fold i ndependent of the expression vector used. In summary, both the 5'UTR a nd 3'UTR of the human TSH receptor mRNA influence the level of recepto r expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the mRNA. These data explain the grea ter level of TSH receptor expression in cell lines that are transfecte d with cDNA lacking these regions of the mRNA transcript.