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

Alisa Boutin,Marvin C Gershengorn,Susanne Neumann,Christine C Krieger,Joanna Klubo-Gwiezdzinska,Bernice Marcus-Samuels

doi:10.3389/fendo.2020.00276

Alisa Boutin, Marvin C Gershengorn + Show 4 more

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https://doi.org/10.3389/fendo.2020.00276

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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 Gi/Go 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.

Highlights

The TSH receptor (TSHR) in human thyroid cells couples to G proteins of all four subfamilies [1], including the stimulatory G protein Gs, which activates adenylyl cyclase to produce cAMP, Gi, which inhibits cAMP production, G13, which activates p44/42 mitogen-activated protein kinase (MAPK) [2], and Gq/G11, which activate phospholipase C to produce inositol-1,4,5trisphosphate, which is rapidly degraded to inositol monophosphate (IP-1; phosphoinositide signaling) [3].TSH receptors (TSHRs) Homodimerization in Human ThyrocytesTSHR oligomerization in primary cultures of thyrocytes and in cells overexpressing TSHRs has been demonstrated by several techniques including fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) [4]
We showed that TSHR expression is much higher in tissue obtained at thyroidectomy than in human thyrocytes (hThyros) in vitro
This possibility underscored the importance of formally testing the hypothesis that in vivo levels of TSHRs were needed for biphasic cAMP regulation

Summary

Introduction

TSHR oligomerization in primary cultures of thyrocytes and in cells overexpressing TSHRs has been demonstrated by several techniques including fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) [4]. BRET experiments by Urizar et al confirmed TSHR homodimerization and demonstrated that the serpentine domain of the TSHR is primarily involved in dimerization [6]. The potency for TSH to stimulate cAMP signaling is ∼100-fold higher than for phosphoinositide signaling [3]. We showed previously that the different potencies occurred because cAMP signaling is initiated by binding of one TSH to one protomer of a putative TSHR homodimer whereas phosphoinositide signaling requires binding of two TSH molecules to the TSHR homodimer [7]

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