A cell line derived from the Fisher rat thyroid (FRT), that does not have functional TSH receptor, was stably transfected with the cDNA of the human TSH receptor (h TSH-R). In wild FRT cells TSH (1-1000 mU/l) was unable to increase cAMP production, while 10-10000 nmol/l forskolin elicited a 10-30 fold cAMP stimulation. Two of the transfected clones were responsive to TSH in terms of cAMP production. In particular, the FRT-R3 transfected clone showed the highest sensitivity to the hormone with a 10 fold cAMP increase over the basal at 100 mU/l TSH. The Northern blot analysis using a 2.4 kbp cDNA probe for the hTSH-R showed a band corresponding to the mRNA of TSH receptor in FRT-R3 cells, but not in wild FRT cells. In both cell types TSH was ineffective in stimulating growth assayed by 3H-thymidine incorporation into DNA. Hybridization with a probe for thyroperoxidase on polymerase chain reaction products after reverse transcription of mRNA showed that FRT-R3, as well as FRT cells, do not have a transcript for thyroperoxidase. In conclusion, the data reported in this paper show that the insertion of the hTSH-R cDNA in the genome of poorly differentiated rat thyroid cells results in the recovery of TSH-dependent adenylate cyclase, but not other differentiated thyroid cell functions.
Transfection with the cDNA of the human thyrotropin receptor of a poorly differentiated rat thyroid cell line (FRT).
ELISEI, ROSSELLA;PINCHERA, ALDO;ROMEI, CRISTINA;SANTINI, FERRUCCIO;VITTI, PAOLO
1996-01-01
Abstract
A cell line derived from the Fisher rat thyroid (FRT), that does not have functional TSH receptor, was stably transfected with the cDNA of the human TSH receptor (h TSH-R). In wild FRT cells TSH (1-1000 mU/l) was unable to increase cAMP production, while 10-10000 nmol/l forskolin elicited a 10-30 fold cAMP stimulation. Two of the transfected clones were responsive to TSH in terms of cAMP production. In particular, the FRT-R3 transfected clone showed the highest sensitivity to the hormone with a 10 fold cAMP increase over the basal at 100 mU/l TSH. The Northern blot analysis using a 2.4 kbp cDNA probe for the hTSH-R showed a band corresponding to the mRNA of TSH receptor in FRT-R3 cells, but not in wild FRT cells. In both cell types TSH was ineffective in stimulating growth assayed by 3H-thymidine incorporation into DNA. Hybridization with a probe for thyroperoxidase on polymerase chain reaction products after reverse transcription of mRNA showed that FRT-R3, as well as FRT cells, do not have a transcript for thyroperoxidase. In conclusion, the data reported in this paper show that the insertion of the hTSH-R cDNA in the genome of poorly differentiated rat thyroid cells results in the recovery of TSH-dependent adenylate cyclase, but not other differentiated thyroid cell functions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
