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Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization

Nature Neuroscience volume 8, pages 881–888 (2005)Cite this article

Abstract

Neurotransmitter transporters are critical for synaptic neurotransmitter inactivation. Transporter inhibitors markedly increase the duration and magnitude of synaptic transmission, underscoring the importance of transporter activity in neurotransmission. Recent studies indicate that membrane trafficking dynamically governs neuronal transporter cell-surface presentation in a protein kinase C–regulated manner, suggesting that transporter trafficking profoundly affects synaptic signaling. However, the molecular architecture coupling neurotransmitter transporters to the endocytic machinery is not defined. Here, we identify nonclassical, distinct endocytic signals in the dopamine transporter (DAT) that are necessary and sufficient to drive constitutive and protein kinase C–regulated DAT internalization. The DAT internalization signal is conserved across SLC6 neurotransmitter carriers and is functional in the homologous norepinephrine transporter, suggesting that this region is likely to be the endocytic signal for all SLC6 neurotransmitter transporters. The DAT endocytic signal does not conform to classic internalization motifs, suggesting that SLC6 neurotransmitter transporters may have evolved unique endocytic mechanisms.

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Figure 1: The DAT N terminus does not contain an autonomous endocytic signal.
Figure 2: DAT C-terminal residues 587–596 are sufficient to drive Tac endocytosis.
Figure 3: DAT Ile595 within the FREKLAYAIA sequence is necessary for Tac-DAT endocytosis.
Figure 4: Nonpolar residues within the FREKLAYAIA sequence are necessary for constitutive DAT internalization as shown by DAT internalization assay.
Figure 5: The FREKLAYAIA region is conserved in SLC6 neurotransmitter transporters and is both necessary and sufficient for NET endocytosis.
Figure 6: Nonpolar residues within the FREKLAYAIA sequence are not required for PKC-stimulated DAT downregulation and internalization.
Figure 7: The DAT C terminus contains a PKC-sensitive endocytic signal.
Figure 8: DAT C-terminal residues 587–596 are sufficient and necessary to drive endocytosis in dopaminergic cells.

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Acknowledgements

We thank Z. Stevens for excellent technical assistance and D. LaPointe for bioinformatics support. We are grateful to V. Budnik, P. Gardner and D. Lambright for critical manuscript review and helpful discussions. This work was supported by National Institute on Drug Abuse award 15169 to H.E.M.

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Authors and Affiliations

  1. Department of Psychiatry University of Massachusetts Medical School, Brudnick Neuropsychiatric Research Institute, 303 Belmont Street, Worcester, 01604, Massachusetts, USA

    Katherine L Holton, Merewyn K Loder & Haley E Melikian

  2. Program in Neuroscience University of Massachusetts Medical School, 303 Belmont Street, Worcester, 01604, Massachusetts, USA

    Haley E Melikian

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 303 Belmont Street, Worcester, 01604, Massachusetts, USA

    Haley E Melikian

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Correspondence to Haley E Melikian.

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Holton, K., Loder, M. & Melikian, H. Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization. Nat Neurosci 8, 881–888 (2005). https://doi.org/10.1038/nn1478

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