Cellular and Molecular Life Sciences

, Volume 75, Issue 12, pp 2227–2239 | Cite as

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

  • Jana Fischer
  • Gunnar Kleinau
  • Claudia Rutz
  • Denise Zwanziger
  • Noushafarin Khajavi
  • Anne Müller
  • Maren Rehders
  • Klaudia Brix
  • Catherine L. Worth
  • Dagmar Führer
  • Heiko Krude
  • Burkhard Wiesner
  • Ralf Schülein
  • Heike Biebermann
Original Article
First Online:
Received:
Revised:
Accepted:

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.

Keywords

GPCR MCT8 MFS transporters Oligomerization TSHR 

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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Notes

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.

Supplementary material

18_2017_2728_MOESM1_ESM.docx (628 kb)
Supplementary material 1 (DOCX 628 kb)

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  • Jana Fischer
    • 1
  • Gunnar Kleinau
    • 1
    • 2
  • Claudia Rutz
    • 3
  • Denise Zwanziger
    • 4
  • Noushafarin Khajavi
    • 1
  • Anne Müller
    • 1
  • Maren Rehders
    • 5
  • Klaudia Brix
    • 5
  • Catherine L. Worth
    • 6
  • Dagmar Führer
    • 4
  • Heiko Krude
    • 1
  • Burkhard Wiesner
    • 3
    • 7
  • Ralf Schülein
    • 3
  • Heike Biebermann
    • 1
  1. 1.Institut für Experimentelle Pädiatrische EndokrinologieCharité – Universitätsmedizin BerlinBerlinGermany
  2. 2.Group Protein X-ray Crystallography and Signal Transduction, Institute of Medical Physics and BiophysicsCharité – Universitätsmedizin BerlinBerlinGermany
  3. 3.Protein Trafficking GroupLeibniz-Forschungsinstitut für Molekulare PharmakologieBerlinGermany
  4. 4.Division of Laboratory Research, Department of Endocrinology, Diabetology and MetabolismUniversity Hospital Essen, University Duisburg-EssenEssenGermany
  5. 5.Department of Life Sciences and ChemistryJacobs University BremenBremenGermany
  6. 6.Structural Bioinformatics and Protein Design GroupLeibniz-Forschungsinstitut für Molekulare PharmakologieBerlinGermany
  7. 7.Cellular Imaging GroupLeibniz-Forschungsinstitut für Molekulare PharmakologieBerlinGermany

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