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The human thyrotropin beta-subunit gene was isolated and characterized from two genomic libraries and found to contain three exons separated by two introns of 3.9 and 0.45 kilobase pairs. Exons 2 and 3 in the mouse thyrotropin beta-subunit gene are not found in humans due to a lack of consensus sequences important in exon splicing. Moreover, using primer extension, RNA sequencing, and S1 nuclease analysis, we determined, in a thyrotropin-producing pituitary adenoma, that exon 1 in humans contains only one transcriptional start site and is 37 base pairs in length. This is unlike both the rat and mouse thyrotropin beta-subunit genes which contain two transcriptional start sites. Changes in the genomic structure of the more 5' "TATA box" and surrounding "CAAT box" might explain why the more 5' start site in humans is apparently not utilized. Moreover, the first exon in humans is longer than the corresponding exon in murine species presumably due to a 9-base pair insertion between the TATA box and transcriptional start site (37 versus 27 nucleotides). Thus, while alternative exon splicing and differential start site utilization in response to thyroid hormone may be important in the regulation of murine thyrotropin beta-subunit genes, they are not found in man.

Mouse pituitary thyrotropic tumor minces were labeled with [35S]methionine and fractionated into rough microsomes, intermediate, and low density smooth microsomes. Thyroid-stimulating hormone subunits were mainly in rough microsomes after a 10-min pulse, but with increasing chase times the proportion in smooth microsomes increased. In rough microsomes, small amounts of an alpha subunit precursor of Mr = 11,000 and larger amounts of an alpha form of Mr = 18,000 were rapidly processed to a form of Mr = 21,000, while small amounts of a beta-subunit precursor of M r = 11,000 were processed to a form of Mr = 18,000. Most of the Mr = 18,000 and Mr = 21,000 subunit forms were converted by endoglycosidase H to forms of Mr = 11,000 to 12,000. Small amounts of endoglycosidase H-resistant forms appeared in low density smooth microsomes after a 30-min chase. Subunit combination was not detected at 10 min; combination was first detected at 20 min and increased progressively to a maximum of 61% of beta in the low density smooth microsomes at 60 min of chase. Although alpha of Mr = 11,000 and 18,000, and beta of Mr - 11,000, were not detected in thyroid-stimulating hormone, both endoglycosidase H-sensitive and -resistant alpha subunit of Mr = 21,000 and beta subunit of Mr = 18,000 were found combined. Thus, the rough endoplasmic reticulum (ER) contains only small amounts of nonglycosylated subunits (Mr = 11,000). The major subunit precursors contain one high mannose oligosaccharide (Mr = 18,000), with a second unit being added onto alpha in the rough ER. Combination of alpha (Mr = 21,000) with beta (Mr = 18,000) begins in the rough ER but occurs predominantly in the smooth ER/Golgi. Oligosaccharides of both combined and uncombined subunits are processed from high mannose to complex forms predominantly in the smooth ER/Golgi.

We have studied the regulation of the biosynthesis of thyrotropin (TSH) and its alpha and beta subunits by thyroid hormone in thyrotropic tumors carried in hypothyroid mice. Treatment with 3,5,3'-triiodo-L-thyronine (T3) (20 micrograms/100 g, body weight) daily for 4 or 10 days reduced serum TSH to 3 and 0.3% of control, respectively. Serum levels of free alpha subunit were reduced to 60 and 11% of control at 4 days and 10 days, respectively, and serum free TSH-beta was undetectable at both time points. There was no significant decrease in tumor TSH content after 4 days of treatment and, after 10 days, TSH content was reduced to 15% of control levels. There was no significant effect of T3 on tumor alpha subunit levels at either 4 or 10 days. In contrast, tumor TSH-beta content was markedly reduced after 4 days and 10 days of T3 treatment, to 29 and 10% of control levels, respectively. Translation of tumor poly(A) mRNA in a rabbit reticulocyte lysate system showed that thyroid hormone decreased translatable TSH-beta mRNA to undetectable levels at both 4 and 10 days, whereas translatable alpha mRNA was reduced strikingly only at 10 days in one of two tumors. RNA blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs showed that TSH-beta mRNA was reduced to less than 10% of control after both 4 and 10 days of T3 treatment, whereas, again, alpha mRNA was only reduced in one of two tumors at 10 days. Our data thus show that thyroid hormone affects alpha and TSH-beta mRNA and protein levels discordantly and suggest that regulation of TSH biosynthesis may occur predominantly at the level of TSH-beta mRNA.