Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mamalian cells.
The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investigated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by deleting either the 5' untranslated region (UTR), the 3'UTR, or both UTRs. The 5'UTR contains two false AUG initiation codons followed by a stop 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 were studied in order to minimize insertion site-dependent variation in the level of expression. Scatchard analysis of TSH binding indicated that the number of receptors on the surface of Chinese hamster ovary cells expressing the wild-type transcript (approximately 16,000/cell) increased approximately 2-fold with 5'UTR deletion, approximately 5-fold with 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 of 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 stimulation was commensurate with the number of receptors expressed on the surface of the different cell lines. Truncation of the 5'UTR did not alter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as well as the 5'3'UTR-deleted transcript, increased approximately 4-fold independent of the expression vector used. In summary, both the 5'UTR and 3'UTR of the human TSH receptor mRNA influence the level of receptor expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the MRNA. These data explain the greater level of TSH receptor expression in cell lines that are transfected with cDNA lacking these regions of the mRNA transcript.
Kakinuma
A
A
Thyroid Molecular Biology Unit, Veterans' Administration Medical Center, San Francisco, California 94121, USA.
Chazenbalk
G
G
Filetti
S
S
McLachlan
S M
SM
Rapoport
B
B
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003062:Codon; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D013972:Thyrotropin / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D003062:Codon; D017931:DNA Primers; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP