Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

Abstract

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587-596 region, although whether PKCinduced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKCstimulated DAT endocytosis. Alanine substituting DAT residues 587-590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588-590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587-590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates.

Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane

“…Similarly, the mutation of Lys 590 and Asp 600 impaired hDAT cell surface expression due to its retention in the ER, and the G585A substitution completely blocked hDAT export from the ER (50). Finally, the central motif FREKLAYAIA (residues 587-596) in hDAT has been suggested to be essential for the constitutive internalization of the transporter (22,(42)(43). Thus, in all of these studies the potential involvement of the initial residues of DAT-CT in trafficking mechanisms was not described nor hypothesized.…”

“…DAT-Ctr9 complex formation appears to be dependent only interaction with the first 24 amino acids of DAT-CT (Tyr 574 -Glu 597 ). Interestingly, this first half of DAT-CT contains a motif (FREKLAYAIA) that was determined to be essential for constitutive DAT-mediated and PKC-stimulated internalization processes (22,42,43). The disruption of this motif in the ⌬32-CT mutant suppressed this interaction.…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Further studies showed that Ala substitutions of residues 587 to 590 were sufficient to abolish PKC-mediated DAT downregulation and constitutive DAT internalization. On the basis of these results, it was suggested that the stretch of four residues is part of an endocytic braking mechanism that is relieved upon PKC stimulation (Boudanova et al, 2008). The role of this C-terminal motif in ubiquitination of the N terminus in PKC-mediated DAT endocytosis has not been elucidated and awaits further investigations.…”

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

“…Studies from our laboratory (23) and others (24) indicate that a unique negative regulatory mechanism, or "endocytic brake," stabilizes DAT surface expression. PKC activation releases the endocytic brake, accelerates DAT internalization, and thereby reduces DAT surface levels and function.…”