• Adenylate cyclase assay
• FlAsH assay
• follicle-stimulating hormone (FHS) receptor (FSHr)
• G-protein coupled receptors (GPCRs)
• luteinizing hormone (LH) receptor (LHr)
• thyrotropin (TSH) receptor (TSHr)
• trichloro- bis(p-chlorophenyl)ethane (DDT)

In the first part of this study, we aimed at establishing whether two previously identified thyroid disruptors, the insecticide 1,1,1-trichloro-2,2- bis(p-chlorophenyl)ethane (DDT) and Aroclor 1254 (a complex mixture of polychlorinated water), may inhibit the thyrotropin (TSH) receptor (TSHr) activity. DDT and Aroclor 1254 were shown to inhibit both the basal and bovine TSH (bTSH)-stimulated accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with the TSHr. Furthermore, both DDT and Aroclor 1254 did indeed prevent cAMP accumulation, as induced by the constitutive activity of a point mutant TSHr(A623I) transiently transfected in African green monkey kidney fibroblast COS-7 cells. Neither trypsin digestion of the extracellular domain (ECD) nor deletion of the ECD in a mutant TSHr trunk transiently transfected in COS-7 cells counteracted the inhibitory activity of DDT and Aroclor 1254. DDT exerted a weak inhibitory activity against forskolin in both CHO-K1 and COS-7 cells, whereas it was nil against the agonists dopamine and 5-(N-ethyl-carboxamido)-adenosine (NECA) in CHO cells stably transfected with the dopamine D1 receptor and in COS-7 cells transiently transfected with the adenosine type 2a receptor (A2a receptor). Furthermore, DDT was inactive against the stimulation by isoproterenol of the endogenously expressed β2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells, but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr. The following experiments was performed to test whether other compounds that are structurally correlated to DDT may also behave as potential TSH receptor inhibitors.
Amongst the compounds structurally homologous to DDT, quercentin, kanferol and stirbestrol showed the strongest inhibitory effect on cAMP accumulation in TSH-stimulated CHO-TSHr transfected cells. Since several of these compounds, like DDT itself, have an estrogenic activity, it is likely that their cell binding sites may be physiologically relevant for estrogens. As a matter of fact, thyroid stimulating hormone (TSH), luteinizing hormone (LH) and follicle-stimulating hormone (FHS) receptors are all members of the glycoprotein hormone receptor family and there are indications that estrogens can inhibit the activity of both LHr and/or FSHr. In line with this view, there are a number of functional experiments showing that DDT may inhibiting the activity of TSHr, LHr and FSHr. Furthermore, the estradiol can also inhibit the LHr activity at concentrations that are considered physiological in the in ovarian follicle, thus indicating that it might play a role in oocyte maturation. These experiments support the concept that DDT may act through an hypothetical “estrogenic binding site” conserved throughout evolution in the glycoprotein hormone receptor family.
To further deepen our knowledge on the effects induced by DDT, we examined how DDT alter the process of TSHr internalization in CHO-TSHr transfected cells. When these cells were probed immunocytochemically with anti-TSH receptor antibodies, the receptor appeared to be mostly expressed on the plasma membrane. By comparison, the overall antibody fluorescence became primarily restricted to a juxta-nuclear position when CHO-TSHr cells were exposed to [1 mU/ml] TSH for 1 or 5 min. However, in the presence of DDT, this fluorescence maintained a peripheral location along the cell plasma membrane, even when CHO-TSHr cells were simultaneously stimulated with TSH for 1 and 5 minutes. To verify the specificity of DDT on TSH receptor, CHO cells transfected with the A2a receptor were used as a control. Following 1 min stimulation with the agonist Neca, A2a receptors were gradually internalized regardless of the presence of DDT in the culture medium, thus confirming the specificity of DDT for the TSHr.
In the last part of the project, we aimed at setting up an assay to perform intramolecular fluorescence resonance energy transfer (FRET) to studying directly the mechanism by which DDT and its analogues interact with the TSHr. To this purpose, a tetracystein motif acting as a binding site for the fluorescein Arsenical Hairpin (FlAsH) compound was inserted into the carboxyl terminal domain of the TSHr. FlAsH is known to become fluorescent only when bound to this tetracystein motif. Due to the small size of the FlAsH coumpound, this modified TSH receptor may prove more adequate for intramolecular FRET than other chromophores that are known to cause large steric hindrance or even interact with the receptor function (GFP, YFP, etc). In this study the TSHr-FlAsH mutantion was initially characterized in several cell lines, and eventually the best results were obtained with HeLa cells. It is my intention to continue these experiments by trying to introduce the FlAsH motif in the third intracellular loop of the TSHr together with the cyan fluorescent protein at its carboxyl terminal. In principle, these modifications should allow us to perform an intramolecular FRET of the TSHr and hopefully to investigate in more detail the effect exerted by estradiol on the LH receptor.