Regulation of G Protein-Coupled Receptors by Ubiquitination.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and ß-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

Characterization of the interaction between the dopamine D4 receptor, KLHL12 and ß-arrestins.

Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as ß-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that ß-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and ß-arrestins and questioned its functional relevance. Our data demonstrate that ß-arrestin 1 and ß-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both ß-arrestins but has no influence on their ubiquitination. Knockout of one of the two ß-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by ß-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation.

Dopamine D4 receptor ubiquitination.

Ubiquitination is a post-translational modification that targets proteins for degradation but can also regulate other cellular processes such as endocytosis, trafficking and DNA repair. We investigate ubiquitination of the dopamine D4receptor (D4R) which belongs to the superfamily of G protein-coupled receptors (GPCR). Several polymorphic variants of the D4R exist, which differ in the number of 16-amino acid repeats in the third intracellular loop (IC3) of the receptor. The functional role of this polymorphic region is not known but persons with the seven-repeat allele show a predisposition to develop attention deficit hyperactivity disorder (ADHD). We identified a protein, KLHL12, which specifically interacts with this polymorphic region and enhances ubiquitination of the D4R. We have tested the influence of KLHL12 on the ubiquitination of the most common D4R polymorphic variants and found that KLHL12 strongly promotes ubiquitination of the two- and four-repeat variant but has hardly any effect on ubiquitination of the seven-repeat D4R. This suggests that differential ubiquitination of the D4R may have functional implications. Moreover, we were able to demonstrate that KLHL12-mediated D4R ubiquitination does not lead to receptor degradation. Next, we aimed to identify specific residues in the sequence of D4R which undergo ubiquitination and observed that the lysine-less receptor mutant is still ubiquitinated. Subsequently, we have tested the hypothesis whether KLHL12 could promote ubiquitination on non-lysine residues of the D4R. The importance of the cysteine and serine/threonine residues in the ubiquitination process of the receptor was examined and the obtained results confirmed that D4R can be ubiquitinated on non-lysine residues. In this review we summarize our data on D4R ubiquitination and put this in the light of other GPCR ubiquitination studies.

KLHL12 Promotes Non-Lysine Ubiquitination of the Dopamine Receptors D4.2 and D4.4, but Not of the ADHD-Associated D4.7 Variant.

DOPAMINE D4 RECEPTOR POLYMORPHISM: The dopamine D4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder. KLHL12 PROMOTES UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR ON NON-LYSINE RESIDUES: In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D4 receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible. DIFFERENTIAL UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR POLYMORPHIC VARIANTS: Additionally, we show that the dopamine D4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D4.2 and D4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process.

Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.

The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.

Role of dimerization in dopamine D(4) receptor biogenesis.

Dopamine receptors are G protein-coupled receptors critically involved in locomotion, reward, and cognitive processes. Export of dopamine receptors to the plasma membrane is thought to follow the default secretory pathway, whereby proteins travel from the endoplasmatic reticulum (ER), through the Golgi apparatus, to arrive at the cell surface. Several observations indicate that trafficking from the ER to the plasma membrane is tightly regulated, and that correct folding in the ER acts as a bottle neck to the maturation of the dopamine D4 receptors. The dopamine D(4) receptor is an interesting receptor since it has a polymorphic region in its third intracellular loop, resulting in receptor isoforms of varying length and amino acid composition. Correct folding is enhanced by: (1) interaction with specific proteins, such as ER resident chaperones, (2) interaction with pharmacological chaperones, for example, ligands that are membrane permeable and can bind to the receptor in the ER, and (3) receptor dimerization; the assembly of multisubunit proteins into a quaternary structure is started in the ER before cell surface delivery, which helps in correct folding and subsequent expression. These interactions help the process of GPCR folding, but more importantly they ensure that only properly folded proteins proceed from the ER to the trans-Golgi network. In this review we will mainly focus on the role of receptor dimerization in dopamine D(4) receptor maturation.

The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components.

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html.

Detection of G protein-coupled receptor (GPCR) dimerization by coimmunoprecipitation.

With 356 members in the human genome, G protein-coupled receptors (GPCRs) constitute the largest family of proteins involved in signal transduction across biological membranes. GPCRs are integral membrane proteins featuring a conserved structural topology with seven transmembrane domains. By recognizing a large diversity of hormones and neurotransmitters, GPCRs mediate signal transduction pathways through their interactions with both extracellular small-molecule ligands and intracellular G proteins to initiate appropriate cellular signaling cascades. As there is a clear link between GPCRs and several disorders, GPCRs currently constitute the largest family of proteins targeted by marketed pharmaceuticals. Therefore, a detailed understanding of the biogenesis of these receptors and of GPCR-protein complex assembly can help to answer some important questions. In this chapter, we will discuss several methods to isolate GPCRs and to study, via coimmunoprecipitation, protein-protein interactions. Special attention will be given to GPCR dimerization, which often starts already in the endoplasmic reticulum and influences the maturation of the receptor. Next, we will also explain an elegant tool to study GPCR biogenesis based on the glycosylation pattern of the receptor of interest.

Dopamine D4 receptor oligomerization--contribution to receptor biogenesis.

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.

KLHL12-mediated ubiquitination of the dopamine D4 receptor does not target the receptor for degradation.

In previous studies, we identified KLHL12 as a novel interaction partner of the dopamine D4 receptor that functions as an adaptor in a Cullin3-based E3 ubiquitin ligase complex to target the receptor for ubiquitination. In this study, we show that KLHL12 promotes poly-ubiquitination of the receptor by performing ubiquitination assays in eukaryotic cells. Furthermore, we demonstrate 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. Unexpectedly, however, KLHL12-mediated receptor ubiquitination 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 ubiquitination or degradation, in contrast to many other G-protein-coupled receptors (GPCRs) indicating that ubiquitination of GPCRs does not defaultly lead to receptor degradation. Interestingly, KLHL12 does also interact with beta-arrestin2 but this has no effect on the ubiquitination or localization of beta-arrestin2 nor on the internalization of the D4 receptor.

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation.

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling. Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor. We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of beta-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor. These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.