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The role of KLHL12 in the signalling and the regulation of the dopamine D4 receptor

Pieter Rondou UGent (2009)
abstract
Dopamine receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) and are subdivided in D1-type (D1, D5) and D2-type (D2, D3, D4) receptors. The human D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated, among others, with Attention Deficit Hyperactivity Disorder (ADHD). We demonstrated that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. Although KLHL12 binds to and promotes ubiquitination of the three main polymorphic D4 receptor variants (D4.2, D4.4 and D4.7), ubiquitination levels decrease with increasing repeat length. In addition, we showed that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors, and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity towards the D4 receptor. Furthermore, we demonstrated that KLHL12 not only interacts with both immature, ER-associated and mature, plasma membrane-associated D4 receptors, but also promotes ubiquitination of both receptor subpools. However, KLHL12 does not promote proteasomal degradation of newly synthesized receptors through the ER-associated degradation pathway or lysosomal degradation of mature receptors. Moreover, our data reveal that D4 receptors do not undergo agonist-promoted internalization, ubiquitination or degradation, in contrast to many other G-protein-coupled receptors (GPCRs). Together, our data show that KLHL12-mediated ubiquitination of the D4 receptor does not target the receptor for degradation, suggesting that D4 receptor ubiquitination, and maybe ubiquitination of GPCRs in general, might have alternative functions. Finally, preliminary data suggest that KLHL12 might instead influence the signalling properties of the D4 receptor.
Please use this url to cite or link to this publication:
author
promoter
Guy Haegeman
organization
year
type
dissertation (monograph)
subject
keyword
ubiquitination, ADHD, D4 receptor, Dopamine, GPCR, KLHL12
pages
257 + addendum pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Wetenschappen (auditorium 1.04, K.L. Ledeganckstraat 35)
defense date
2009-09-11 16:00
language
English
UGent publication?
yes
classification
D1
copyright statement
I have retained and own the full copyright for this publication
id
751372
handle
http://hdl.handle.net/1854/LU-751372
alternative location
http://lib.ugent.be/fulltxt/RUG01/001/376/245/RUG01-001376245_2010_0001_AC.pdf
date created
2009-09-21 16:37:08
date last changed
2009-10-14 12:14:37
@phdthesis{751372,
  abstract     = {Dopamine receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) and are subdivided in D1-type (D1, D5) and D2-type (D2, D3, D4) receptors. The human D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated, among others, with Attention Deficit Hyperactivity Disorder (ADHD). We demonstrated that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. Although KLHL12 binds to and promotes ubiquitination of the three main polymorphic D4 receptor variants (D4.2, D4.4 and D4.7), ubiquitination levels decrease with increasing repeat length. In addition, we showed that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors, and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity towards the D4 receptor. Furthermore, we demonstrated that KLHL12 not only interacts with both immature, ER-associated and mature, plasma membrane-associated D4 receptors, but also promotes ubiquitination of both receptor subpools. However, KLHL12 does not promote proteasomal degradation of newly synthesized receptors through the ER-associated degradation pathway or lysosomal degradation of mature receptors. Moreover, our data reveal that D4 receptors do not undergo agonist-promoted internalization, ubiquitination or degradation, in contrast to many other G-protein-coupled receptors (GPCRs). Together, our data show that KLHL12-mediated ubiquitination of the D4 receptor does not target the receptor for degradation, suggesting that D4 receptor ubiquitination, and maybe ubiquitination of GPCRs in general, might have alternative functions. Finally, preliminary data suggest that KLHL12 might instead influence the signalling properties of the D4 receptor.},
  author       = {Rondou, Pieter},
  keyword      = {ubiquitination,ADHD,D4 receptor,Dopamine,GPCR,KLHL12},
  language     = {eng},
  pages        = {257 + addendum},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {The role of KLHL12 in the signalling and the regulation of the dopamine D4 receptor},
  url          = {http://lib.ugent.be/fulltxt/RUG01/001/376/245/RUG01-001376245\_2010\_0001\_AC.pdf},
  year         = {2009},
}

Chicago
Rondou, Pieter. 2009. “The Role of KLHL12 in the Signalling and the Regulation of the Dopamine D4 Receptor”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Rondou, P. (2009). The role of KLHL12 in the signalling and the regulation of the dopamine D4 receptor. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Rondou P. The role of KLHL12 in the signalling and the regulation of the dopamine D4 receptor. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2009.
MLA
Rondou, Pieter. “The Role of KLHL12 in the Signalling and the Regulation of the Dopamine D4 Receptor.” 2009 : n. pag. Print.