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ABSTRACT: Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.
Journal of Biological Chemistry 05/2008; 283(16):10978-91. · 4.77 Impact Factor
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ABSTRACT: Different Na+/Cl--dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular
compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist
as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution
and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization
at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase
treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface.
Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that
was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial
homolog LeuTAa, as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed
to contribute to the dimerization interface of GlyTs.
Journal of Biological Chemistry 04/2008; 283(16):10978-10991. · 4.77 Impact Factor
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ABSTRACT: The human dopamine transporter (hDAT) contains a C-terminal type 2 PDZ (postsynaptic density 95/Discs large/zona occludens 1) domain-binding motif (LKV) known to interact with PDZ domain proteins such as PICK1 (protein interacting with C-kinase 1). As reported previously, we found that, after deletion of this motif, hDAT was retained in the endoplasmic reticulum (ER) of human embryonic kidney (HEK) 293 and Neuro2A cells, suggesting that PDZ domain interactions might be critical for hDAT targeting. Nonetheless, substitution of LKV with SLL, the type 1 PDZ-binding sequence from the beta2-adrenergic receptor, did not disrupt plasma membrane targeting. Moreover, the addition of an alanine to the hDAT C terminus (+Ala), resulting in an LKVA termination sequence, or substitution of LKV with alanines (3xAla_618-620) prevented neither plasma membrane targeting nor targeting into sprouting neurites of differentiated N2A cells. The inability of +Ala and 3xAla_618-620 to bind PDZ domains was confirmed by lack of colocalization with PICK1 in cotransfected HEK293 cells and by the inability of corresponding C-terminal fusion proteins to pull down purified PICK1. Thus, although residues in the hDAT C terminus are indispensable for proper targeting, PDZ domain interactions are not required. By progressive substitutions with beta2-adrenergic receptor sequence, and by triple-alanine substitutions in the hDAT C terminus, we examined the importance of epitopes preceding the LKV motif. Substitution of RHW(615-617) with alanines caused retention of the transporter in the ER despite preserved ability of this mutant to bind PICK1. We propose dual roles of the hDAT C terminus: a role independent of PDZ interactions for ER export and surface targeting, and a not fully clarified role involving PDZ interactions with proteins such as PICK1.
Journal of Neuroscience 09/2004; 24(31):7024-36. · 7.11 Impact Factor
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ABSTRACT: Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with the extracellular end of transmembrane segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted DAT (CD-DAT) into which only Cys243 or Cys306 was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were cross-linked at a symmetrical dimer interface. Reintroduction of both Cys243 and Cys306 into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2-12 and other DAT inhibitors, including benztropine and mazindol, protected Cys243 against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking. We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4 interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure of the transporter.
Journal of Biological Chemistry 12/2003; 278(46):45045-8. · 4.77 Impact Factor
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ABSTRACT: Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with
the extracellular end of transmembrane segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility
that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands
on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted
DAT (CD-DAT) into which only Cys243 or Cys306 was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were
cross-linked at a symmetrical dimer interface. Reintroduction of both Cys243 and Cys306 into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer
bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in
the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2–12 and other
DAT inhibitors, including benztropine and mazindol, protected Cys243 against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking.
We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4
interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure
of the transporter.
Journal of Biological Chemistry 11/2003; 278(46):45045-45048. · 4.77 Impact Factor
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ABSTRACT: Increasing evidence suggests that Na(+)/Cl(-)-dependent neurotransmitter transporters exist as homo-oligomeric proteins. However, the functional implication of this oligomerization remains unclear. Here we demonstrate the engineering of a Zn(2+) binding site at the predicted dimeric interface of the dopamine transporter (DAT) corresponding to the external end of transmembrane segment 6. Upon binding to this site, which involves a histidine inserted in position 310 (V310H) and the endogenous Cys306 within the same DAT molecule, Zn(2+) potently inhibits [(3)H]dopamine uptake. These data provide indirect evidence that conformational changes critical for the translocation process may occur at the interface between two transporter molecules in the oligomeric structure.
FEBS Letters 08/2002; 524(1-3):87-91. · 3.54 Impact Factor
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ABSTRACT: Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.
Molecular Pharmacology 08/2002; 62(1):30-7. · 4.88 Impact Factor
