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Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.

ABSTRACT: The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, but the cells still exhibited 100-fold differences in potencies. Dose responses for TSH-stimulated dissociation of prebound (125)I-TSH (negative cooperativity; EC(50)=70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH-stimulated IP1 production (EC(50)=50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding-deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH-stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G(q/11).

SUBMITTER: Allen MD

PROVIDER: S-EPMC3177577 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.

Allen Michael D MD Neumann Susanne S Gershengorn Marvin C MC

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20110624 10

The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, ...[more]

PMID: 21705666

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Project description:Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7??M. The selection criteria for a positive hit were based on a mean signal threshold of ?50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3??M and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in GD.

Dataset Information

Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.

Publications

Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.

Similar Datasets

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