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Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7(a) receptor

Evelien Gellynck UGent, Kjetil W Andressen, Béatrice Lintermans UGent, Guy Haegeman UGent, Finn O Levy, Peter Vanhoenacker and Kathleen Van Craenenbroeck UGent (2012) FEBS JOURNAL. 279(11). p.1994-2003
abstract
The 5-hydroxytryptamine (5-HT)7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [3H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT7 receptor.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
QUALITY-CONTROL, SPLICE VARIANTS, IDENTIFICATION, PROTEIN, 5-HT4(B), OLIGOSACCHARIDES, DOPAMINE-RECEPTORS, CELL-SURFACE EXPRESSION, SITE-DIRECTED MUTAGENESIS, PLASMA-MEMBRANE LOCALIZATION, serotonin receptor, N-glycosylation, membrane trafficking, G-protein-coupled receptor
journal title
FEBS JOURNAL
FEBS J.
volume
279
issue
11
pages
1994 - 2003
Web of Science type
Article
Web of Science id
000304041600008
JCR category
BIOCHEMISTRY & MOLECULAR BIOLOGY
JCR impact factor
4.25 (2012)
JCR rank
74/288 (2012)
JCR quartile
2 (2012)
ISSN
1742-464X
DOI
10.1111/j.1742-4658.2012.08581.x
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
2913898
handle
http://hdl.handle.net/1854/LU-2913898
date created
2012-06-21 09:17:42
date last changed
2012-07-05 16:01:09
@article{2913898,
  abstract     = {The 5-hydroxytryptamine (5-HT)7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [3H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT7 receptor.},
  author       = {Gellynck, Evelien and Andressen, Kjetil W and Lintermans, B{\'e}atrice and Haegeman, Guy and Levy, Finn O and Vanhoenacker, Peter and Van Craenenbroeck, Kathleen},
  issn         = {1742-464X},
  journal      = {FEBS JOURNAL},
  keyword      = {QUALITY-CONTROL,SPLICE VARIANTS,IDENTIFICATION,PROTEIN,5-HT4(B),OLIGOSACCHARIDES,DOPAMINE-RECEPTORS,CELL-SURFACE EXPRESSION,SITE-DIRECTED MUTAGENESIS,PLASMA-MEMBRANE LOCALIZATION,serotonin receptor,N-glycosylation,membrane trafficking,G-protein-coupled receptor},
  language     = {eng},
  number       = {11},
  pages        = {1994--2003},
  title        = {Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7(a) receptor},
  url          = {http://dx.doi.org/10.1111/j.1742-4658.2012.08581.x},
  volume       = {279},
  year         = {2012},
}

Chicago
Gellynck, Evelien, Kjetil W Andressen, Béatrice Lintermans, Guy Haegeman, Finn O Levy, Peter Vanhoenacker, and Kathleen Van Craenenbroeck. 2012. “Biochemical and Pharmacological Study of N-linked Glycosylation of the Human Serotonin 5-HT7(a) Receptor.” Febs Journal 279 (11): 1994–2003.
APA
Gellynck, E., Andressen, K. W., Lintermans, B., Haegeman, G., Levy, F. O., Vanhoenacker, P., & Van Craenenbroeck, K. (2012). Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7(a) receptor. FEBS JOURNAL, 279(11), 1994–2003.
Vancouver
1.
Gellynck E, Andressen KW, Lintermans B, Haegeman G, Levy FO, Vanhoenacker P, et al. Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7(a) receptor. FEBS JOURNAL. 2012;279(11):1994–2003.
MLA
Gellynck, Evelien, Kjetil W Andressen, Béatrice Lintermans, et al. “Biochemical and Pharmacological Study of N-linked Glycosylation of the Human Serotonin 5-HT7(a) Receptor.” FEBS JOURNAL 279.11 (2012): 1994–2003. Print.