Small-molecule thyrotropin receptor agonist activates naturally occurring thyrotropin-insensitive mutants and reveals their distinct cyclic adenosine monophosphate signal persistence

Abstract

Background: Subclinical hypothyroidism (SHT), characterized by normal thyroid hormone levels maintained by elevated thyrotropin (TSH), predisposes patients to health problems as they age. Some cases arise from mutations of the TSH receptor (TSHR) that confer TSH resistance. This resistance might be circumvented by TSHR agonists with different modes of binding compared with TSH. We hypothesized that the recently discovered small-molecule TSHR agonist C2, with its unique mode of receptor binding, would activate mutant TSHRs associated with SHT, facilitating their study.

Materials and methods: HEK-EM293 cells transiently expressing TSHR variants-wild-type TSHR or mutants C41S, L252P, L467P, or C600R-were analyzed for TSH or C2-induced cyclic adenosine monophosphate (cAMP) signaling to establish C2 as a mutant TSHR agonist. These cells were also pretreated with TSH or C2 to characterize each mutant receptor's ability to maintain and desensitize cAMP signaling.

Results: We showed that C2 could activate the TSH-unresponsive TSHR ectodomain mutants C41S and L252P but had no effect on the serpentine mutant L467P. We found that TSH and C2 could acutely activate the serpentine mutant C600R. Preincubation with C2 caused persistent cAMP signaling and receptor desensitization in wild-type TSHR and cAMP signal persistence with no detectable desensitization in the cases of C41S and L252P.

Conclusions: The small-molecule agonist C2 is a useful pharmacological tool for the study of mutant TSHRs. It revealed that some naturally occurring TSH-insensitive mutants can mediate induction of cAMP elevation upon stimulation with C2 and that this signal is differentially maintained within cells.

FIG. 1.

Expression profiles of thyrotropin receptor (TSHR) variants. HEK-EM293 cells were transfected with plasmids encoding the listed TSHR variants. After 48 hours, all were probed with 2C11 or 4C1 antibody, both specific to two different epitopes within the extracellular portion of TSHR. All variants showed reduced cell surface presentation compared with wild-type TSHR. Bars represent mean ± standard error of the mean (SEM).

FIG. 2.

Ligand sensitivity profiles of TSHR variants: TSHR (A), C600R (B), L467P (C), C41S (D), and L252P (E). Transfected HEK-EM293 cells were stimulated with DMSO, 10 mU/mL TSH, or 10 μM C2 for 1 hour in the presence of 500 μM 3-isobutyl-1-methylxanthine (IBMX). After incubation with agonists, intracellular cyclic adenosine monophosphate (cAMP) was measured and three distinct profiles arose. Bars represent mean ± SEM. **p ≤ 0.005.

FIG. 3.

Dose–response curves to TSHR variants TSHR (A), C600R (B), C41S (C), and L252P (D) to TSH and C2. Cells were stimulated for 1 hour in the presence of IBMX. All cAMP levels were normalized to the maximal TSH response from cells expressing TSHR. Please note the varying amplitudes of the y-axes. Bars represent mean ± SEM.

FIG. 4.

C2 causes persistent cAMP signaling and desensitization of TSHR (A) and C600R (B). Cells were pretreated with DMSO, TSH, or C2 for 20 hours and then incubated without ligands for 24 hours. Bars represent mean ± SEM. *p ≤ 0.05; **p ≤ 0.005.

FIG. 5.

Responses of pretreated TSHR variants C41S and L252P. Transfected HEK-EM293 cells were pretreated with DMSO or C2 (30 μM) for 18–20 hours. After an additional 24-hour recovery period, the cells were stimulated with DMSO or C2, and then intracellular cAMP levels were measured. TSHR (A) does show persistent signaling as well as desensitization, C41S (B), and L252P (C) show a strong persistence in cAMP signal. Bars represent mean ± SEM. Please note the varying amplitudes of the y-axes. *p ≤ 0.05; **p ≤ 0.005.