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Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor

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Abstract

The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

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Abbreviations

GPHR:

Glycoprotein hormone receptor

LHCGR:

Lutropin/choriogonadotropin receptor

FSHR:

Follicle stimulating hormone receptor

TSHR:

Thyroid stimulating hormone receptor

bTSH:

Bovine thyroid stimulating hormone

LH:

Luteinizing hormone

CG:

Choriogonadotropin

FSH:

Follicle stimulating hormone

GPCR:

G-protein-coupled receptor

TMH:

Transmembrane helix

ECL1/2/3:

Extracellular loops 1/2/3

ICLs 1/2/3:

Intracellular loops 1/2/3

LRRD:

Leucine-rich repeat domain

SD:

Serpentine domain

CAM:

Constitutively activating mutation

LGRs:

Leucine-rich repeat containing GPCRs

7TMRs:

Seven-transmembrane spanning receptors

c52:

Compound 52

org41841:

Organon 41841

DMEM:

Dulbecco’s modified Eagle’s medium

PBS:

Phosphate buffered saline

IP:

Inositol phosphate

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (KR1273/4-1) and by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

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Authors and Affiliations

  1. Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str.10, 13125, Berlin, Germany

    Ann-Karin Haas, Gunnar Kleinau, Inna Hoyer, Jens Furkert, Claudia Rutz, Ralf Schülein & Gerd Krause

  2. NIDDK, National Institutes of Health Clinical Endocrinology Branch, Bethesda, MD, 20892, USA

    Susanne Neumann & Marvin C. Gershengorn

Authors
  1. Ann-Karin Haas
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  2. Gunnar Kleinau
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  3. Inna Hoyer
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  4. Susanne Neumann
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  5. Jens Furkert
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  6. Claudia Rutz
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  8. Marvin C. Gershengorn
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  9. Gerd Krause
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Correspondence to Gerd Krause.

Additional information

A.-K. Haas and G. Kleinau contributed equally to this work.

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Haas, AK., Kleinau, G., Hoyer, I. et al. Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor. Cell. Mol. Life Sci. 68, 159–167 (2011). https://doi.org/10.1007/s00018-010-0451-2

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