The thyroid-stimulating hormone (TSH) receptor (TSHr) was made specifically fluorescent by insertion of a tetracysteine motif (TSHr-FlAsH) into the C terminal end and transiently transfected into COS-7 and HeLa cells. The observation that TSH administration caused the intracellular level of cAMP to increase in both TSHr-FlAsH-transfected cell types indicated that the FlAsH binding motif did not alter normal TSHr functioning. When transfected into HeLa cells and stimulated with TSH, the TSHr-FlAsH receptor exhibited a pronounced perinuclear labelling pattern, whereas labelling remained on the cell surface following pre-incubation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT). Chinese hamster ovary (CHO)-TSHr cells probed with anti-TSHr antibodies were fluorescent mainly in the proximity of the plasma membrane, with fluorescence being primarily restricted to a juxta-nuclear position when exposed to 10 mU/ml TSH for 1 or 5 min. However, in the presence of DDT, the anti-TSHr fluorescence maintained a peripheral location along the cell plasma membrane, even if CHO-TSHr cells were stimulated with TSH for 1 and 5 min. To verify that DDT acted specifically on the TSHr, CHO cells transfected with the A2a receptor were used as controls. Following a 1-min stimulation with 5’- (N-ethyl-carboxamido)-adenosine, A2a receptors were gradually internalized regardless of the presence of DDT in the culture medium. Finally, immunoelectron microscopy of CHO-TSHr cells showed that a 1-min exposure to TSH sufficed to displace anti-TSHr antibodies tagged with 10-nm gold particles into coated pits and vesicles but that their superficial location was retained along the plasma membrane in the presence of DDT.
Thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) prevents internalization of TSH receptor
PELLEGRINO, MARIO;
2009-01-01
Abstract
The thyroid-stimulating hormone (TSH) receptor (TSHr) was made specifically fluorescent by insertion of a tetracysteine motif (TSHr-FlAsH) into the C terminal end and transiently transfected into COS-7 and HeLa cells. The observation that TSH administration caused the intracellular level of cAMP to increase in both TSHr-FlAsH-transfected cell types indicated that the FlAsH binding motif did not alter normal TSHr functioning. When transfected into HeLa cells and stimulated with TSH, the TSHr-FlAsH receptor exhibited a pronounced perinuclear labelling pattern, whereas labelling remained on the cell surface following pre-incubation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT). Chinese hamster ovary (CHO)-TSHr cells probed with anti-TSHr antibodies were fluorescent mainly in the proximity of the plasma membrane, with fluorescence being primarily restricted to a juxta-nuclear position when exposed to 10 mU/ml TSH for 1 or 5 min. However, in the presence of DDT, the anti-TSHr fluorescence maintained a peripheral location along the cell plasma membrane, even if CHO-TSHr cells were stimulated with TSH for 1 and 5 min. To verify that DDT acted specifically on the TSHr, CHO cells transfected with the A2a receptor were used as controls. Following a 1-min stimulation with 5’- (N-ethyl-carboxamido)-adenosine, A2a receptors were gradually internalized regardless of the presence of DDT in the culture medium. Finally, immunoelectron microscopy of CHO-TSHr cells showed that a 1-min exposure to TSH sufficed to displace anti-TSHr antibodies tagged with 10-nm gold particles into coated pits and vesicles but that their superficial location was retained along the plasma membrane in the presence of DDT.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
