title: The role of N-glycosylation in function and surface trafficking of the human dopamine transporter.
abstract: The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.
authors.affiliation: Department of Biological Sciences, Illinois State University, Normal, IL 61790, USA.
authors.forename: Li-Bin
authors.initials: LB
authors.lastname: Li
authors.forename: Nianhang
authors.initials: N
authors.lastname: Chen
authors.forename: Sammanda
authors.initials: S
authors.lastname: Ramamoorthy
authors.forename: Limen
authors.initials: L
authors.lastname: Chi
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Cui
authors.forename: Lijuan C
authors.initials: LC
authors.lastname: Wang
authors.forename: Maarten E A
authors.initials: ME
authors.lastname: Reith
mesh_terms: D019943:Amino Acid Substitution; D019921:Biotinylation; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D018492:Dopamine Antagonists / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D018492:Dopamine Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C493214:SLC6A3 protein, human; D014415:Tunicamycin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase