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Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level

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Abstract

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

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Abbreviations

BiFc:

Bimolecular fluorescence complementation

FCCS:

Fluorescence cross-correlation spectroscopy

MCT8:

Mono-carboxylate transporter 8

TSHR:

Thyrotropin receptor

GPCR:

G-protein-coupled receptor

MFS:

Major facilitator superfamily

smTIRF:

Single molecule total internal reflection microscopy

AT1R:

Angiotensin-1-receptor (AT1R)

TRPC3:

Transient receptor potential cation channel subfamily C3

rM3R:

Muscarinic acetylcholine receptor 3 (rat)

MC4R:

Melanocortin receptor 4

ELISA:

Enzyme linked immunosorbent assay

cAMP:

Cyclic adenosine mono phosphate

IP3 :

Inositol triphosphate

T3:

3,3,5-triiodo-l-thyronine

TH:

Thyroid hormone

TSH:

Thyroid stimulating hormone

YFP:

Yellow fluorescent protein

GD:

Graves disease

FA:

Follicular adenoma

FTC:

Follicular thyroid carcinoma

PTC:

Papillary thyroid carcinoma

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), projects BI 893/6-3, KL 2334/2-2, in the framework of the SPP 1629 “Thyroid Trans Act” BI893/5-2, BR 1308/11-2, KR1710/5-1, ZW 221/2-1, FU356/3-3 and FU356/7-2. We would like to acknowledge the assistance of the BCRT Flow Cytometry Lab (Charité-Universitätsmedizin Berlin). We would also like to thank Sabine Jyrch, Cigdem Cetindag and Jenny Eichhorst for excellent technical assistance, Vahid Asimi for independent reproduction of the FCCS data and Martin Lehmann for useful discussions.

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Authors and Affiliations

  1. Institut für Experimentelle Pädiatrische Endokrinologie, Charité – Universitätsmedizin Berlin, 13353, Berlin, Germany

    Jana Fischer, Gunnar Kleinau, Noushafarin Khajavi, Anne Müller, Heiko Krude & Heike Biebermann

  2. Group Protein X-ray Crystallography and Signal Transduction, Institute of Medical Physics and Biophysics, Charité – Universitätsmedizin Berlin, 10117, Berlin, Germany

    Gunnar Kleinau

  3. Protein Trafficking Group, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Claudia Rutz, Burkhard Wiesner & Ralf Schülein

  4. Division of Laboratory Research, Department of Endocrinology, Diabetology and Metabolism, University Hospital Essen, University Duisburg-Essen, 45147, Essen, Germany

    Denise Zwanziger & Dagmar Führer

  5. Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759, Bremen, Germany

    Maren Rehders & Klaudia Brix

  6. Structural Bioinformatics and Protein Design Group, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125, Berlin, Germany

    Catherine L. Worth

  7. Cellular Imaging Group, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125, Berlin, Germany

    Burkhard Wiesner

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  1. Jana Fischer
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  2. Gunnar Kleinau
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Corresponding authors

Correspondence to Ralf Schülein or Heike Biebermann.

Additional information

Jana Fischer, Gunnar Kleinau denotes equal contributions.

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Fischer, J., Kleinau, G., Rutz, C. et al. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level. Cell. Mol. Life Sci. 75, 2227–2239 (2018). https://doi.org/10.1007/s00018-017-2728-1

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