Subcellular redistribution of the serotonin transporter by secretory carrier membrane protein 2.
The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.
Müller
Heidi Kaastrup
HK
School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin 4, Ireland, Trinity College Dublin 2, Ireland.
Wiborg
Ove
O
Haase
Jana
J
D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins / Q000096:biosynthesis*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D008565:Membrane Proteins; C466776:SCAMP2 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C106354:flotillins; D005982:Glutathione Transferase