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Membrane Glycoprotein M6B Interacts with the Human Serotonin Transporter

Abstract

The serotonin transporter (SERT) belongs to a family of sodium- and chloride-dependent neurotransmitter transporters that are responsible for the active re-uptake of the neurotransmitter serotonin from the synapse. In the present study, using the yeast two-hybrid system, we identified the membrane glycoprotein M6B as a binding partner of SERT. This interaction was further verified by co-immunoprecipitation and glutathione-S-transferase pull-down assays. M6B belongs to a proteolipid protein family, which is expressed in neurons and in oligodendrocytes in the brain. The knowledge of the biological function of this protein family is sparse, but their expression in most brain regions have led to the hypothesis that they are involved in cellular housekeeping functions such as membrane trafficking and cell-to-cell communication. The co-expression of SERT with M6B results in a significant decrease in SERT-mediated serotonin uptake caused by a down-regulation of SERT surface expression. Furthermore, we find, using confocal microscopy, that M6B co-localizes with SERT when transiently expressed in HEK-MSR-293 cells and when endogenously expressed in RN46A cells. Taken together, our data suggest that M6B regulates the serotonin uptake by affecting cellular trafficking of the serotonin transporter.

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Abbreviations

SERT:

serotonin transporter

DAT:

dopamine transporter

NET:

norephinephrine transporter

5-HT:

serotonin

SCAMP-2:

secretory carrier associated membrane protein

PLP:

proteolipid protein

TM1:

transmembrane domain 1

PBS:

phosphate-buffered saline

HEK-MSR-293:

human embryonic kidney-293

RN46A:

immortalized raphe nucleus cells

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Acknowledgments

The work was partly carried out during an extended stay by A.W. Fjorback in the lab of Jana Haase, School of Biomolecular and Biomedical Sciences, Conway Institute, University of College Dublin, Ireland; we are grateful for her supervision and assistance. We thank Karina Birk Nygaard for her technical assistance. We are grateful to Dr. H. Werner, for providing antibodies and the M6B plasmid, and to Dr. Narayanan, for providing the M6A plasmids. This work was supported by the EU Marie Curie Molecular Neuroimmunology PhD Programme (MNIEST, contract number MEST-CT-514333), UCD Conway Institute, Dublin, Ireland and Eli Lilly Foundation, Denmark.

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Correspondence to Ove Wiborg.

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Fjorback, A.W., Müller, H.K. & Wiborg, O. Membrane Glycoprotein M6B Interacts with the Human Serotonin Transporter. J Mol Neurosci 37, 191–200 (2009). https://doi.org/10.1007/s12031-008-9092-4

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  • DOI: https://doi.org/10.1007/s12031-008-9092-4

Keywords

  • Interacting protein
  • Yeast two-hybrid
  • GST pull-down
  • Co-immunoprecipitation
  • Surface expression
  • Confocal microscopy
  • Cellular trafficking