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. 2015 Jan 1;25(1):51–62. doi: 10.1089/thy.2014.0119

FIG. 4.

FIG. 4.

TSHR signaling via different G proteins. In order to study the engagement of different G proteins with the TSHR, we generated stable CHO cells containing various luciferase reporter constructs with distinct response elements as detailed in Materials and Methods. (A) Upon stimulation of the TSHR, the most predominant activation occurred through Gsα, which lead to an increase in cAMP via activation of adenylyl cyclase. The cAMP increase ultimately results in the activation of the CRE fused to the luciferase gene. As shown, molecules MS437 and MS438 were potent stimulators of cAMP generation via activation of Gsα (**p<0.001). (B) Similarly, a stable line of CHO cells co-transfected with the TSHR and an NFAT response element-containing luciferase construct in order to measure signaling through Gαq showed that the two lead molecules were capable of activating this G protein to the same extent as 1000 μIU/mL of TSH or, as control, 1 μM ionomycin+10 ng/mL Phorbal-12-myristate-13-actate (PMA; **p<0.01). (C) SRF-RE is a response element fused to the luciferase reporter gene and is able to measure Gα12 activation via enhancement of the second messenger Rho A kinase. The data indicate that the molecules are able to engage this pathway as was both 20% serum and TSH, which acted as positive controls (**p<0.01). (D) Activation of Gβγ leads to activation of second messenger ERK1/2, which in turn can activate the SRE-RE. This graph shows that the cells are responsive to 20% serum with 10 ng/mL of Phorbol-12-myristate-13-acetate (PMA) as a positive control. No responses were seen with TSH or MS437 and MS438 (**p<0.01). (E) Rat thyrocytes (FRTL5) grown to near confluence in 6H medium were maintained for another 48 h without TSH. Prior to RNA extraction, the cells were treated with TSH 1000 μIU/mL or 10 μM of MS437 at 37°C for 4 h. The different panels indicate the fold changes in the expression of mRNA for Tg (top panel), NIS (middle panel), and the TSHR (lower panel) in the presence of medium only, TSH, or molecule MS437. Small molecule MS437 increased the expression anywhere between two- and sixfold for each of the genes assayed (**p<0.01).