http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Tue, 21 May 2013 09:38:59 GMT 2013-05-21T09:38:59Z http://hdl.handle.net/2067/1561 Title: The thyroid disruptor 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) prevents internalization of the TSH receptor Authors: Picchietti, Simona; Belardinelli, Maria Cristina; Taddei, Anna Rita; Fausto, Anna Maria; Pellegrino, M.; Rossi, M.; Maggio, Roberto; Giorgi, Franco 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. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1561 2008-12-31T23:00:00Z