Ubiquitination of CXCR7 Controls Receptor Trafficking

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gαi-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process

The viral G protein-coupled receptor ORF74 hijacks β-arrestins for endocytic trafficking in response to human chemokines

Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74

Photoswitching the Efficacy of a Small-Molecule Ligand for a Peptidergic GPCR: from Antagonism to Agonism

For optical control of GPCR function, we set out to develop small-molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene-containing CXCR3 ligands. G protein activation assays and real-time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron-donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling

A toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with light

We report a detailed structure–activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle efficacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H₁ Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

The histamine H(1) receptor (H(1)R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H(1)R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H(1)R signals via heterotrimeric G(q) proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H(1)R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H(1)R signaling through heterotrimeric G(q) proteins, second messengers (inositol 1,4,5-triphosphate and Ca(2+)), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H(1)R over G(q) biosensor activation

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H1R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H1R signals via heterotrimeric Gq proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H1R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H1R signaling through heterotrimeric Gq proteins, second messengers (inositol 1,4,5-triphosphate and Ca2+), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H1R over Gq biosensor activation

ORF74 internalizes and traffics via early, recycling and late endosomes.

<p>HEK293T cells were transiently transfected with ORF74-Rluc8 (WT) (A-D) or ORF74-ST/A2-Rluc8 (ST/A2) (E-H) in combination with Venus-K-Ras (plasma membrane marker) (A, E), Venus-Rab5a (early endosome marker) (B, F), Venus-Rab7a (late endosome/lysosome marker) (C, G) or Venus-Rab11 (recycling endosome marker) (D, H) and stimulated with CXCL1, CXCL8 or CXCL10 for indicated time and BRET was measured. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over vehicle-stimulated cells (dotted line) and error bars indicate SEM values. Statistical differences between the area under the curve of vehicle- and corresponding CXCL1-, CXCL8- or CXCL10-treated cells (baseline = 1) were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001, *** p≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.</p

Characterization and β-arrestin recruitment to ORF74-R^3.50A.

<p>(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R^3.50A (R^3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R^3.50A-Rluc8 (R^3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R^3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).</p

ORF74 recruits β-arrestin1 and β-arrestin2 in response to human chemokines.

<p>HEK293T cells co-expressing ORF74-Rluc8 and β-arrestin1-eYFP (A, C) or β-arrestin2-eYFP (B, D) were stimulated with increasing concentrations of CXCL1 (open squares), CXCL8 (filled squares) or CXCL10 (open circles) (A, B) or co-stimulated with CXCL1 and CXCL10 (C, D). β-arrestin recruitment to the receptor was measured as an increase in BRET ratio (BRETr). Data are shown as fold over basal and represent the mean of pooled data from at least three independent experiments each performed in triplicate. Error bars indicate SEM values. Significant differences between vehicle and chemokine-stimulation were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001). NS = not significant.</p

Characterization and β-arrestin recruitment to ORF74-ST/A.

<p>(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine in ORF74-ST/A are shown in bold brown. The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124486#pone.0124486.ref035" target="_blank">35</a>]. (B-F) HEK293T cells were transiently transfected with WT-ORF74 (WT) (B-E) or ORF74-ST/A (ST/A) (B-F) or empty vector DNA (mock-transfected) (B, E). (B) Relative receptor expression at the cell surface was determined by ELISA. Binding of ¹²⁵I-CXCL10 (C) or ¹²⁵I-CXCL8 (D) to intact HEK293T cells was measured in the presence of increasing concentrations unlabeled homologous chemokines. Constitutive (E) or chemokine-induced (F) activation of PLC was determined by measuring InsP accumulation. (G) HEK293T cells expressing ORF74-Rluc8 (WT) or ORF74-ST/A-Rluc8 (ST/A) in combination with β-arrestin1-eYFP (βarr1) or β-arrestin2-eYFP (βarr2) were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) before measurement of BRET. Data are presented as fold over mock-transfected cells (dotted line) (B, E), percentage of specific binding (C, D) or fold over basal (F, G). All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (B) or constitutive PLC activation (E) between WT-ORF74 and ORF74-ST/A or between vehicle- and corresponding CXCL1-treated cells (G) were determined by a Student t test (**** p ≤ 0.0001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.</p