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Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, ?-arrestin translocation and chemotaxis assays.

ABSTRACT: Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells.We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [(125)I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through ?-arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter ?-arrestin translocation.There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G-protein-dependent and -independent functional responses.Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of ?-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization.

SUBMITTER: Gilchrist A

PROVIDER: S-EPMC4253460 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays.

Gilchrist A A Gauntner T D TD Fazzini A A Alley K M KM Pyen D S DS Ahn J J Ha S J SJ Willett A A Sansom S E SE Yarfi J L JL Bachovchin K A KA Mazzoni M R MR Merritt J R JR

British journal of pharmacology 20141101 22

<h4>Background and purpose</h4>Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to ...[more]

PMID: 24990525

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Project description:BackgroundThe human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8). It is involved in inflammatory diseases as well as cancer. Consequently, much effort is put into the identification of CXCR2 targeting drugs. Fundamental research regarding CXCR2 signaling is mainly focused on CXCL8 (IL-8), which is the first and best described high-affinity ligand for CXCR2. Much less is known about CXCR2 activation induced by other chemokines and it remains to be determined to what extent potential ligand bias exists within this signaling system. This insight might be important to unlock new opportunities in therapeutic targeting of CXCR2.MethodsLigand binding was determined in a competition binding assay using labeled CXCL8. Activation of the ELR + chemokine-induced CXCR2 signaling pathways, including G protein activation, β-arrestin1/2 recruitment, and receptor internalization, were quantified using NanoBRET-based techniques. Ligand bias within and between these pathways was subsequently investigated by ligand bias calculations, with CXCL8 as the reference CXCR2 ligand. Statistical significance was tested through a one-way ANOVA followed by Dunnett's multiple comparisons test.ResultsAll chemokines (CXCL1-3 and CXCL5-8) were able to displace CXCL8 from CXCR2 with high affinity and activated the same panel of G protein subtypes (Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gα15) without any statistically significant ligand bias towards any one type of G protein. Compared to CXCL8, all other chemokines were less potent in β-arrestin1 and -2 recruitment and receptor internalization while equivalently activating G proteins, indicating a G protein activation bias for CXCL1,-2,-3,-5,-6 and CXCL7. Lastly, with CXCL8 used as reference ligand, CXCL2 and CXCL6 showed ligand bias towards β-arrestin1/2 recruitment compared to receptor internalization.ConclusionThis study presents an in-depth analysis of signaling bias upon CXCR2 stimulation by its chemokine ligands. Using CXCL8 as a reference ligand for bias index calculations, no ligand bias was observed between chemokines with respect to activation of separate G proteins subtypes or recruitment of β-arrestin1/2 subtypes, respectively. However, compared to β-arrestin recruitment and receptor internalization, CXCL1-3 and CXCL5-7 were biased towards G protein activation when CXCL8 was used as reference ligand.

Project description:PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Project description:Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

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Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, ?-arrestin translocation and chemotaxis assays.

Publications

Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays.

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