Biomedical subjects

R L Warne

Publications and source records attributed to R L Warne.

4 recordsLinked to original sources

PubMed1992

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Affinity Labels↗

PubMed1989

Mutations of the rat growth hormone promoter which increase and decrease response to thyroid hormone define a consensus thyroid hormone response element.

We have previously identified sequences required for thyroid hormone (T3) induction of the rat GH (rGH) promoter, which lie in a region from -188 to -164 upstream of the mRNA start site. Within this region, Domains A, -189 to -184 and B, -179 to -174, are imperfect direct repeats, and domain C, -172 to -167, is a divergent inverted copy that matches the A domain at 4/6 positions. A series of synthetic mutant versions of this sequence were inserted upstream of a truncated rGH promoter, or as a replacement for wild-type sequences in a synthetic 237 base pair rGH promoter or upstream of the heterologous thymidine kinase promoter. Mutations changing the B domain to a perfect copy of the A domain significantly increased T3 induction (21.3-fold) relative to the wild type (3.6-fold). A single point mutation making the C domain a better match to the A domain also increased T3 induction to 16.2-fold. Combining this up-mutation with any of three down-mutations in the A, B, or C domains strongly decreased response, showing that all three domains contribute to the amplified T3 response. Binding affinity of the various mutant oligonucleotides was assessed using in vitro translated receptor and affinity paralleled the functional responses for most binding site mutations. Requirements for in vitro binding were, however, less rigorous than those for functional T3 induction. Based on these results, we propose a consensus T3 receptor binding half-site, AGGT(C/A)A, at least two copies of which are required for a T3 response.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1988

Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.

Amino Acid Sequence↗

PubMed1987

Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone.

Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. We have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. We show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor.

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