TSH Receptor Homodimerization in Regulation of cAMP Production in Human Thyrocytes in vitro

Abstract

Thyrotropin hormone (TSH) was reported to exhibit biphasic regulation of cAMP production in human thyroid slices; specifically, upregulation at low TSH doses transitioning to inhibition at high doses. We observed this phenomenon in HEK293 cells overexpressing TSH receptors (TSHRs) but in only 25% of human thyrocytes (hThyros) in vitro. Because TSHR expression in hThyros in vitro was low, we tested the hypothesis that high, in situ levels of TSHRs were needed for biphasic cAMP regulation. We increased expression of TSHRs by infecting hThyros with adenoviruses expressing human TSHR (AdhTSHR), measured TSH-stimulated cAMP production and TSHR homodimerization. TSHR mRNA levels in hThyros in vitro were 100-fold lower than in human thyroid tissue. AdhTSHR infection increased TSHR mRNA expression to levels found in thyroid tissue and flow cytometry showed that cell-surface TSHRs increased more than 15-fold. Most uninfected hThyro preparations exhibited monotonic cAMP production. In contrast, most hThyro preparations infected with AdhTSHR expressing TSHR at in vivo levels exhibited biphasic TSH dose responses. Treatment of AdhTSHR-infected hThyros with pertussis toxin resulted in monotonic dose response curves demonstrating that lower levels of cAMP production at high TSH doses were mediated by G_i/G_o proteins. Proximity ligation assays confirmed that AdhTSHR infection markedly increased the number of TSHR homodimers. We conclude that in situ levels of TSHRs as homodimers are needed for hThyros to exhibit biphasic TSH regulation of cAMP production.

Keywords: TSHR; cAMP production; inverted U-shaped dose response curve; phosphoinositide signaling; receptor homodimerization; thyrotropin receptor.

Figure 1

Comparison of the levels of TSHR mRNA in thyroid tissue and hThyros in vitro. There were 29 samples of thyroid tissue and 31 samples of hThyros. The mRNA levels were normalized to GAPDH mRNA. The values were markedly different (P <0.0001).

Figure 2

TSHR mRNA and protein expression in hThyros infected with AdhTSHR. (A) Effect of increasing AdhTSHR MOI on the level of TSHR mRNA in hThyros in culture. The measurements were performed in thyrocytes from four different donors in duplicate. Data are expressed as mean ± SD. (B) hThyros infected with increasing amounts of AdhTSHR were analyzed for KSAb1-647 fluorescence by flow cytometry. Bars depict mean KSAb1-647 fluorescence of labeled cells. Representative data from one thyrocyte strain are shown, and this experiment was repeated in thyrocytes from four different donors. Increase of TSHR protein expression following infection with 50 MOI was donor dependent and ranged from 5 to 63-fold over control (0 MOI).

Figure 3

In vivo level expression of TSHR is required for biphasic cAMP production induced by TSH in hThyros. Cells were uninfected (0 MOI) or infected with AdhTSHR at 1, 10, and 50 MOI. Cells were not exposed to TSH (Basal) or were stimulated with TSH at 1 or 100 mU/ml. cAMP production was measured in cells incubated in buffer containing IBMX to inhibit cAMP degradation. Results shown are from 2 experiments performed in triplicate and are expressed as mean ± SEM. We observed statistically significant increase in constitutive levels of cAMP with increasing MOI of AdhTSHR (P <0.01 between 0 and 1 MOI and P <0.0001 between 0 and 10 and 50 MOI). The effects of 100 mU/ml TSH were reduced compared to those of 1 mU/ml at 10 and 50 MOI (P <0.045 between 1 and 100 mU/ml with 10 MOI and P <0.01 between 1 and 100 mU/ml with 50 MOI) with the decrease in cAMP greater at AdhTSHR of 50 MOI than at 10 MOI (P <0.04).

Figure 4

Biphasic cAMP production is stimulated by low TSH doses and inhibited by G_i/G_o at high TSH doses. hThyros were infected with AdhTSHR at 1 MOI (squares) or 50 MOI (circles). Pertussis toxin (100 ng/ml) was added to cells 18 h prior to adding increasing doses of TSH. cAMP production was measured in cells incubated in buffer containing IBMX to inhibit cAMP degradation. Results shown are from 3 experiments performed in duplicate and presented as mean±SEM. In hThyros infected with AdhTSHR at 50 MOI and stimulated with high TSH doses, cAMP production exhibits a classic IUDRC. The decreasing response at 10 and 100 mU/ml TSH is abolished in cells pretreated with pertussis toxin (PTX) (P <0.01 and P <0.005, respectively).

Figure 5

Formation of TSHR homodimers in hThyros infected with AdhTSHR. Increasing protein expression induces the formation of TSHR homodimers. Positive dots (red) in the proximity ligation assay decorate TSHR homodimers. Micrographs represent data from one of three thyrocyte strains from different donors. Cell boundaries were marked using Phalloidin Alexa 633 (green). Cell number was determined by SYTO9 staining (blue). We found no statistically significant difference between 0 and 1 MOI. The differences between uninfected cells and cells infected with 10 and 50 MOI were highly statistically significant (P <0.0001).