G-Protein-Coupled Receptor Screen Reveals a Role for Chemokine Receptor CCR5 in Suppressing Microglial Neurotoxicity

Abstract M-tropic human immunodeficiency virus (HIV-1) strains enter the cell after interaction with their receptors, CD4 and the G-protein–coupled chemokine receptor CCR5. The number of cell surface CCR5 molecules is thought to be important in determining the infection rate for HIV. Cell surface CCR5 is dependent on the rate of receptor internalization and recycling. Internalization of G-protein–coupled receptors after agonist activation is thought to occur either through clathrin-coated pits or through caveolae. In this study, the role of these different pathways was investigated in Chinese hamster ovary cells expressing CCR5 using specific inhibitors. Internalization of CCR5 after chemokine treatment was inhibited by sucrose, indicating a role for the clathrin-coated pit pathway. Activation of CCR5 leads to arrestin-2 movement in the cells, providing further evidence for the involvement of clathrin-coated pits. Nystatin and filipin also affected the rate of internalization of CCR5, indicating a role for caveolae. Using inhibitors of vesicle transport in the cell, it was found that the CCR5 recycling pathway is independent of the Golgi apparatus and late endosomes. Protein synthesis is not involved in receptor recovery. It seems likely that after internalization, CCR5 is directed to early endosomes and subsequently recycled to the cell surface.

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The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology

Cells ◽

10.3390/cells10010075 ◽

2021 ◽

Vol 10 (1) ◽

pp. 75

Author(s):

Marta Laganà ◽

Géraldine Schlecht-Louf ◽

Françoise Bachelerie

Keyword(s):

G Protein ◽

Chemokine Receptor ◽

Immune Cell ◽

Neutrophil Migration ◽

G Protein Coupled Receptor ◽

Receptor Kinases ◽

Immune Cell Migration ◽

And Migration ◽

G Protein Coupled ◽

Gpcr Desensitization

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

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The Functional Expression Methods of Activated G Protein-Coupled Receptor on the Model of Chemokine Receptor

Seibutsu Butsuri ◽

10.2142/biophys.45.236 ◽

2005 ◽

Vol 45 (5) ◽

pp. 236-242

Author(s):

Yoshitake MAEDA

Keyword(s):

G Protein ◽

Chemokine Receptor ◽

Functional Expression ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Effect of Ligand Binding on the Diffusion and Distribution of the G Protein-Coupled Receptor CCR5 in the Cell Membrane

Biophysical Journal ◽

10.1016/j.bpj.2011.11.2806 ◽

2012 ◽

Vol 102 (3) ◽

pp. 512a-513a

Author(s):

Zuleika Calderin-Sollet ◽

Oliver Hartley ◽

Alexandre Fuerstenberg

Keyword(s):

Cell Membrane ◽

Ligand Binding ◽

G Protein ◽

G Protein Coupled Receptor ◽

G Protein Coupled ◽

Receptor Ccr5

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Selection of Active ScFv to G-Protein-Coupled Receptor CCR5 Using Surface Antigen-Mimicking Peptides†

Biochemistry ◽

10.1021/bi0492152 ◽

2004 ◽

Vol 43 (39) ◽

pp. 12575-12584 ◽

Cited By ~ 22

Author(s):

Ying Zhang ◽

Chadler Pool ◽

Kristen Sadler ◽

He-ping Yan ◽

Jennifer Edl ◽

...

Keyword(s):

G Protein ◽

Surface Antigen ◽

G Protein Coupled Receptor ◽

G Protein Coupled ◽

Mimicking Peptides ◽

Receptor Ccr5 ◽

Selection Of

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Human G Protein–Coupled Receptor Gpr-9-6/Cc Chemokine Receptor 9 Is Selectively Expressed on Intestinal Homing T Lymphocytes, Mucosal Lymphocytes, and Thymocytes and Is Required for Thymus-Expressed Chemokine–Mediated Chemotaxis

Journal of Experimental Medicine ◽

10.1084/jem.190.9.1241 ◽

1999 ◽

Vol 190 (9) ◽

pp. 1241-1256 ◽

Cited By ~ 352

Author(s):

Brian A. Zabel ◽

William W. Agace ◽

James J. Campbell ◽

Heidi M. Heath ◽

David Parent ◽

...

Keyword(s):

Small Intestine ◽

T Lymphocytes ◽

G Protein ◽

Chemokine Receptor ◽

Chemokine Receptors ◽

Intraepithelial Lymphocytes ◽

Mucosal Immune Response ◽

G Protein Coupled Receptor ◽

Cc Chemokine ◽

G Protein Coupled

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein–coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti–GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory α4β7high intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen–positive (CLA+) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic α4β7−CLA− memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti–GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

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Gα Protein Selectivity Determinant Specified by a Viral Chemokine Receptor-Conserved Region in the C Tail of the Human Herpesvirus 8 G Protein-Coupled Receptor

Journal of Virology ◽

10.1128/jvi.78.5.2460-2471.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2460-2471 ◽

Cited By ~ 22

Author(s):

Chaoqi Liu ◽

Gordon Sandford ◽

Guo Fei ◽

John Nicholas

Keyword(s):

Protein Kinase ◽

G Protein ◽

Chemokine Receptor ◽

Human Herpesvirus ◽

Human Herpesvirus 8 ◽

G Protein Coupled Receptor ◽

Herpesvirus 8 ◽

Protein Coupling ◽

Conserved Region ◽

G Protein Coupled

ABSTRACT The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-κB, mitogen-activated protein kinase, and Ca2+ signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Gα protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Gαi (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [35S]GTPγS incorporation assays revealed preferential coupling of vGPCR.15 to Gαq and an inability of vGPCR.8 to couple functionally to Gαq. However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Gαq. Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Gα interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Gα protein coupling specificity.

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