N-linked oligosaccharides are required for cell surface expression of the norepinephrine transporter but do not influence substrate or inhibitor recognition.
The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N-glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order Vmax of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.
Nguyen
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Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201, USA.
Amara
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D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D006031:Glycosylation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D027981:Symporters; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D014415:Tunicamycin; D009638:Norepinephrine