G Protein-coupled Receptors Ubiquitination Research Articles

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

Monitoring GPCR Induced Atypical MAPK p38 Signaling to Identify Key Regulators of Vascular Inflammation

Activation of the mitogen activated protein kinase (MAPK) p38 is a critical regulatory step in pro‐inflammatory signaling and a key target for therapeutic intervention in vascular diseases, including chronic inflammation and lung disease. Our recent studies defined a conserved ubiquitin driven pathway that regulates atypical p38 signaling by a subset of G protein‐coupled receptors (GPCR). Atypical p38 signaling is initiated by GPCR induced c‐Src phosphorylation of an autoinhibitory peptide in the E3 ubiquitin ligase NEDD4‐2. In turn, phospho‐activation releases the autoinhibitory peptide from the catalytic site of NEDD4‐2, a step which is critical for stress induced GPCR ubiquitination and amplification of atypical p38 signaling and inflammation in the vasculature. Despite the critical role of GPCR induced p38 signaling in the onset and progression of many pathophysiological responses, atypical p38 signaling is still relatively understudied. Here we report the development of Försters Resonance Energy Transfer (FRET) biosensors to map the spatiotemporal activation of p38 signaling by multiple GPCRs in vascular endothelial cells. These FRET sensors create an ideal platform for a small‐scale screening pipeline to identify molecular modulators of the atypical p38 signaling pathway. Our studies have already identified multiple regulatory proteins that block the induction and/or magnitude of GPCR induced p38 signaling, perturbing pro‐inflammatory signaling in the vasculature.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Detection of β-Arrestin-Mediated G Protein-Coupled Receptor Ubiquitination Using BRET.

Ubiquitination of G protein-coupled receptors (GPCRs) is an important dynamic posttranslational modification that has been linked to the intracellular trafficking of internalized GPCRs to lysosomes. Ubiquitination of GPCRs is mediated by specific E3 ubiquitin ligases that are scaffolded by the adaptor proteins called β-arrestins. Traditionally, detection of GPCR ubiquitination is achieved by using ubiquitin antibodies to Western blot immunoprecipitates of detergent-solubilized GPCRs expressed in heterologous cells. However, studies have also shown that bioluminescence resonance energy transfer (BRET)-based techniques can reveal ubiquitination of GPCRs in intact cells and in real time. This chapter describes a step-by-step protocol to evaluate ubiquitination of GPCRs using the BRET methodology.

Endo-lysosomal sorting of G-protein-coupled receptors by ubiquitin: Diverse pathways for G-protein-coupled receptor destruction and beyond.

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein-coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo-lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo-lysosomal sorting and beyond.

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.

Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors.

The seven-transmembrane containing G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. Transmembrane signaling by GPCRs is fundamental to many aspects of physiology including vision, olfaction, cardiovascular, and reproductive functions as well as pain, behavior and psychomotor responses. The duration and magnitude of signal transduction is tightly controlled by a series of coordinated trafficking events that regulate the cell-surface expression of GPCRs at the plasma membrane. Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs. Of the various molecular mechanisms that impart selectivity, sensitivity and strength of transmembrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR. Ubiquitination of proteins was originally discovered in the context of lysosome-independent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes. In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling. To date about 40 GPCRs are known to be ubiquitinated. For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes. This chapter will focus on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

Ubiquitination of G Protein-Coupled Receptors: Functional Implications and Drug Discovery

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of signaling receptors and control a vast array of physiological responses. Modulating the signaling responses of GPCRs therapeutically is important for the treatment of various diseases, and discovering new aspects of GPCR signal regulation is critical for future drug development. Post-translational modifications are integral to the regulation of GPCR function. In addition to phosphorylation, many GPCRs are reversibly modified with ubiquitin. Ubiquitin is covalently attached to lysine residues within the cytoplasmic domains of GPCRs by ubiquitin ligases and removed by ubiquitin-specific proteases. In many cases, ubiquitin functions as a sorting signal that facilitates trafficking of mammalian GPCRs from endosomes to lysosomes for degradation, but not all GPCRs use this pathway. Moreover, there are distinct types of ubiquitin conjugations that are known to serve diverse functions in controlling a wide range of cellular processes, suggesting broad roles for GPCR ubiquitination. In this review, we highlight recent studies that illustrate various roles for ubiquitin in regulation of GPCR function. Ubiquitination is known to target many GPCRs for lysosomal degradation, and current studies now indicate that basal ubiquitination, deubiquitination, and transubiquitination of certain GPCRs are important for controlling cell surface expression and cellular responsiveness. In addition, novel functions for ubiquitin in regulation of GPCR dimers and in mediating differential GPCR regulation induced by biased agonists have been reported. We will discuss the implications of these new discoveries for ubiquitin regulation of GPCR function in the context of drug development.

On the track of function of KLHL12-mediated dopamine D4 receptor ubiquitination

Event Abstract Back to Event On the track of function of KLHL12-mediated dopamine D4 receptor ubiquitination Kamila Skieterska1*, Pieter Rondou1, Dasiel O. Borroto-Escuela2, Dorien Clarisse1, Béatrice Lintermans1, Guy Haegeman1, Kjell Fuxe2 and Kathleen Van Craenenbroeck1 1 Ghent University, Physiology, Belgium 2 Karolinska Institutet, Neuroscience, Sweden The dopamine D4 receptor belongs to the superfamily of G protein-coupled receptors (GPCRs), the most common drug targets. The dopamine D4 gene displays a remarkable polymorphisms that differ in a variable number of tandem repeats in the region encoding the third intracellular loop. The most frequent variants in the human population are respectively: the 4-repeat allele (D4.4R), 7-repeat allele (D4.7R) and 2-repeat allele (D4.2R), of which the D4.7R polymorphic variant is associated with a predisposition to develop Attention Deficit Hyperactivity Disorder (ADHD). The functional role of the receptor polymorphic region is still poorly understood. We have identified a BTB-Kelch protein, KLHL12, which specifically interacts with the polymorphic repeats and enhances ubiquitination of the D4R. Interestingly, however, we have found that KLHL12 only marginally affects ubiquitination of the D4.7R variant, in contrast to strong induction of ubiquitination of the D4.2R and D4.4R variants. This suggests that differential ubiquitination of the D4R has functional implications. Most studies have focused on the role of GPCR ubiquitination in receptor degradation; nevertheless, our experimental results gave clear evidence that KLHL12-mediated D4R ubiquitination does not lead to receptor degradation. Moreover, our preliminary data suggest that KLHL12 might mediate a non-lysine ubiquitination of the receptor which is currently studied more profoundly. Furthermore, we are investigating the influence of ubiquitination on receptor signaling and found a role in modulation of cAMP level upon D4R activation. Acknowledgements This work was financially supported by FWO (Fonds voor Wetenschappelijk Onderzoek) Vlaanderen: project No. G.0109.09N. K. Skieterska is a recipient of a pre-doctoral FWO fellowship. K. Van Craenenbroeck has a post-doctoral FWO fellowship. Keywords: dopamine D4 receptor, Ubiquitination, KLHL12, ADHD, GPCR Conference: Belgian Brain Council, Liège, Belgium, 27 Oct - 27 Oct, 2012. Presentation Type: Poster Presentation Topic: Other basic/clinical neurosciences topic Citation: Skieterska K, Rondou P, Borroto-Escuela DO, Clarisse D, Lintermans B, Haegeman G, Fuxe K and Van Craenenbroeck K (2012). On the track of function of KLHL12-mediated dopamine D4 receptor ubiquitination. Conference Abstract: Belgian Brain Council. doi: 10.3389/conf.fnhum.2012.210.00053 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 05 Sep 2012; Published Online: 12 Sep 2012. * Correspondence: Ms. Kamila Skieterska, Ghent University, Physiology, Ghent, 9000, Belgium, kamila.skieterska@ugent.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kamila Skieterska Pieter Rondou Dasiel O Borroto-Escuela Dorien Clarisse Béatrice Lintermans Guy Haegeman Kjell Fuxe Kathleen Van Craenenbroeck Google Kamila Skieterska Pieter Rondou Dasiel O Borroto-Escuela Dorien Clarisse Béatrice Lintermans Guy Haegeman Kjell Fuxe Kathleen Van Craenenbroeck Google Scholar Kamila Skieterska Pieter Rondou Dasiel O Borroto-Escuela Dorien Clarisse Béatrice Lintermans Guy Haegeman Kjell Fuxe Kathleen Van Craenenbroeck PubMed Kamila Skieterska Pieter Rondou Dasiel O Borroto-Escuela Dorien Clarisse Béatrice Lintermans Guy Haegeman Kjell Fuxe Kathleen Van Craenenbroeck Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

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 β-arrestin2 but this has no effect on the ubiquitination or localization of β-arrestin2 nor on the internalization of the D4 receptor.

Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics

Sphingosine 1-phosphate subtype 1 (S1P(1)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P(1) down-regulation. Discovery of the in vivo active S1P(1)-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P(1) function while highlighting differences regarding agonist effect on S1P(1) fate, because SEW2871 does not degrade S1P(1). To further understand the mechanism of agonist-induced S1P(1) down-regulation, we compared signaling and fate of human S1P(1)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P(1) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S1P(1) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P(1). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P(1) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P(1) (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.

Oligomerization of G-Protein-Coupled Receptors: Lessons from the YeastSaccharomyces cerevisiae

G-protein-coupled receptors (GPCR) signaling is an evolutionarily ancient mechanism used by all eukaryotes to sense environmental stimuli and mediate cell-cell communication (7, 28). During evolution, GPCR genes expanded enormously in number and diversity. Whereas only three of the ∼5,900 genes in the yeast Saccharomyces cerevisiae encode GPCRs, at least 55 of the ∼12,000 genes in Dictyostelium encode GPCRs (19), and more than 1,000 of the ∼22,000 genes in humans encode receptors of this class (28). GPCRs are of crucial physiologic importance. In eukaryotic microorganisms, GPCRs regulate cell growth, development, morphogenesis, motility, and life span. In humans they mediate the action of hundreds of peptide hormones, sensory stimuli, autacoids, neurotransmitters, and chemokines. GPCRs also are targets of many clinically important drugs as well as drugs of abuse. Despite exhibiting striking diversity in primary sequence and biologic function, GPCRs possess the same fundamental architecture, consisting of seven transmembrane (TM) domains and share common mechanisms of signal transduction (85). GPCRs transduce extracellular signals by coupling to heterotrimeric guanine nucleotide binding proteins (G proteins) consisting of α, β, and γ subunits. Activated GPCRs stimulate exchange of GTP for GDP on Gα subunits, dissociating Gα and Gβγ subunits that, in turn, trigger biological responses by binding effector proteins that regulate second messenger production, protein kinase cascades, cytoskeletal organization, gene transcription, and ion channel activity. GPCRs also signal by G protein-independent mechanisms through recruitment of scaffold proteins such as β-arrestins (reviewed in reference 54). In the budding yeast S. cerevisiae, GPCR signaling regulates two biologic processes: conjugation and nutrient sensing (reviewed in references 15 and 106). During conjugation, a mating type cells secrete a-factor, a 12-residue farnesylated oligopeptide pheromone that binds the G-protein-coupled a-factor receptor (STE3 gene product) expressed only by cells of the α mating type. Conversely, α cells secrete α-factor, an unmodified 13-residue peptide pheromone that binds the G-protein-coupled α-factor receptor (STE2 gene product) expressed only by cells of the a mating type. Although a- and α-factor receptors are unrelated in primary sequence, they trigger similar intracellular responses by activating the same G protein-linked mitogen-activated protein kinase cascade. Many fundamentally important aspects of GPCR signaling were first elucidated in budding yeast (reviewed in reference 16), including cloning of the first nonsensory GPCR, signaling by Gβγ subunits, GPCR ubiquitination during endocytosis, signaling via mitogen-activated protein kinase cascades and scaffolding proteins, and G protein regulation by RGS proteins. The third GPCR in budding yeast, encoded by the GPR1 gene, is a likely receptor for glucose, sucrose, and possibly other ligands (55). This receptor regulates yeast pseudohyphal differentiation, cell size, and life span (44, 58, 102, 109). The Gpr1 homolog of the pathogenic fungus Candida albicans promotes the yeast-to-hyphae transition (67). Gpr1 in S. cerevisiae signals via a pathway using a classical Gα subunit homolog (113) and novel kelch-repeat proteins (3, 35, 36) but lacking typical Gβγ subunits. Like budding yeast, the fission yeast Schizosaccharomyces pombe possesses three GPCRs (reviewed in reference 41). The mam2+ and mam3+ genes encode receptors for the peptide mating pheromones p-factor and m-factor, respectively, whereas the git3+ gene encodes a putative glucose receptor. In this review we highlight current understanding of GPCR oligomerization revealed by studies of the α-factor receptor of budding yeast. With the exception of Mam2 in fission yeast, GPCR oligomerization in other eukaryotic microorganisms has yet to be investigated.