Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent

Amphetamine (AMPH) is a potent dopamine (DA) transporter (DAT) inhibitor that markedly increases extracellular DA levels. In addition to its actions as a DAT antagonist, acute AMPH exposure induces DAT losses from the plasma membrane, implicating transporter-specific membrane trafficking in amphetamine\u27s actions. Despite reports that AMPH modulates DAT surface expression, the trafficking mechanisms leading to this effect are currently not defined. We recently reported that DAT residues 587-596 play an integral role in constitutive and protein kinase C (PKC)-accelerated DAT internalization. In the current study, we tested whether the structural determinants required for PKC-stimulated DAT internalization are necessary for AMPH-induced DAT sequestration. Acute amphetamine exposure increased DAT endocytic rates, but DAT carboxy terminal residues 587-590, which are required for PKC-stimulated internalization, were not required for AMPH-accelerated DAT endocytosis. AMPH decreased DAT endocytic recycling, but did not modulate transferrin receptor recycling, suggesting that AMPH does not globally diminish endocytic recycling. Finally, treatment with a PKC inhibitor demonstrated that AMPH-induced DAT losses from the plasma membrane were not dependent upon PKC activity. These results suggest that the mechanisms responsible for AMPH-mediated DAT internalization are independent from those governing PKC-sensitive DAT endocytosis

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

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 PKC-induced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKC-stimulated 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

High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain

Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu²⁺ oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ∼2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (∼5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfide-linked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets