RANA-CATESBEIANA TADPOLE RED-BLOOD-CELLS EXPRESS AN ALPHA, BUT NOT A BETA, C-ERBA GENE

Thyroid hormone (TH) receptor number in red blood cells (RBCs) from Rana catesbeiana (RC) tadpoles increases 4-fold during both spontaneous and TH-induced metamorphosis, an effect that we have previously shown to be preceded by an increase in the level of c-erbA-related mRNA. The goals of the present study were to obtain an RC c-erbAalpha cDNA that contains the entire open reading frame for a putative TH receptor protein, to determine if this protein has characteristics typical of a TH receptor, and to assess its contribution to the developmentally related increase in TH receptor number. To accomplish this, the missing 5'-sequence of a previously isolated partial RC c-erbAalpha cDNA (RC12) was synthesized by polymerase chain reaction (PCR) and spliced to RC12 to yield a 1490-basepair cDNA (RC15) that contained the entire coding sequence of the receptor protein. Transcription of RC15 followed by translation of its mRNA in a rabbit reticulolysate system yielded a 50-kilodalton protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds T3 with high affinity (K(d), approximately 0.1 nm), and its affinity for T3 is at least 5 times that for T4. The results of cotransfection studies indicate that RC15 can function as a TH receptor; when COS cells were cotransfected with a construct consisting of RC15 cloned in the expression vector CMV4 and TK28 mult, a construct containing rat GH gene TH response element sequences up-stream of a chloramphenicol acetyltransferase reporter gene, chloramphenicol acetyltransferase activity is expressed in the presence, but not in the absence, of T3. To determine whether RBCs contain any c-erbAbeta mRNA transcripts that might contribute to the developmentally related increase in the transcripts detected using RC c-erbAalpha cDNAs, alpha- and beta-specific cDNAs were synthesized by PCR and used as probes in a variety of hybridization assays. In all experiments using conditions in which c-erbAbeta transcripts were detectable in other tissues, there was no evidence that tadpole RBCs contained such species. Lack of any beta-specific transcripts was confirmed by PCR, using as template cDNA prepared by reverse transcription of RC RBC RNA. It was also noted that the RBC at metamorphic climax is the tissue with the highest content of alpha-specific c-erbA transcripts. It is concluded that the c-erbAalpha gene encodes a TH receptor, and that only the alpha-gene is expressed in tadpole RBCs and subject to regulation during development and by TH.