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Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5.

Bönsch C, Munteanu M, Rossitto-Borlat I, Fürstenberg A, Hartley O - PLoS ONE (2015)

Bottom Line: The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors.This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV.Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva. Geneva, Switzerland.

ABSTRACT
G protein-coupled receptors (GPCRs) are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling). The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

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Related in: MedlinePlus

Ligand-directed post-endocytic sorting of CCR5.A. CHO CCR5 cells growing on coverslips were pre-treated (60 min, 4°C) with 100 nM chemokines and anti-CCR5 antibody 3A9 (green), then washed and incubated at 37°C for the indicated times. Cells were then acid washed to remove cell surface antibody, then fixed, permeabilized, and labeled for the ERC marker Rab11 (red) and DAPI (blue, nuclear staining) prior to analysis by confocal scanning microscopy are shown, scale bar = 20 μm. Throughout the experiment the ERC marker (red) remains in a discrete supranuclear spot that is visble in the center of the nuclei (blue) in the maximum intensity projections. Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to colocalize with the ERC marker. After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker. B. Individual Z-slice images from an identical experiment in which cells incubated with the indicated chemokines for 180 min were also labeled for either the ERC marker, Rab11 or the TGN marker, TGN38 (red). Slices in which the marked compartment is most abundant (through the middle of the nucleus for TGN, just above the nucleus for ERC) were chosen. While in cells treated with PSC-RANTES for 180 min, CCR5 colocalizes with TGN38 and does not colocalize with Rab11, in cells treated with 5P14-RANTES for 180 CCR5 colocalizes with Rab11 and does not colocalize with TGN38 scale bar = 20 μm.
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pone.0125396.g001: Ligand-directed post-endocytic sorting of CCR5.A. CHO CCR5 cells growing on coverslips were pre-treated (60 min, 4°C) with 100 nM chemokines and anti-CCR5 antibody 3A9 (green), then washed and incubated at 37°C for the indicated times. Cells were then acid washed to remove cell surface antibody, then fixed, permeabilized, and labeled for the ERC marker Rab11 (red) and DAPI (blue, nuclear staining) prior to analysis by confocal scanning microscopy are shown, scale bar = 20 μm. Throughout the experiment the ERC marker (red) remains in a discrete supranuclear spot that is visble in the center of the nuclei (blue) in the maximum intensity projections. Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to colocalize with the ERC marker. After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker. B. Individual Z-slice images from an identical experiment in which cells incubated with the indicated chemokines for 180 min were also labeled for either the ERC marker, Rab11 or the TGN marker, TGN38 (red). Slices in which the marked compartment is most abundant (through the middle of the nucleus for TGN, just above the nucleus for ERC) were chosen. While in cells treated with PSC-RANTES for 180 min, CCR5 colocalizes with TGN38 and does not colocalize with Rab11, in cells treated with 5P14-RANTES for 180 CCR5 colocalizes with Rab11 and does not colocalize with TGN38 scale bar = 20 μm.

Mentions: A full list of antibodies used in this study is provided in Table 1. For live microscopy, the anti-CCR5 monoclonal antibody Hek/1/85a (AB_369016, Serotec) was rhodamine-labeled using amine-reactive N-hydroxy-succinimidyl-Rhodamine (Pierce) according to the manufacturer’s instructions.


Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5.

Bönsch C, Munteanu M, Rossitto-Borlat I, Fürstenberg A, Hartley O - PLoS ONE (2015)

Ligand-directed post-endocytic sorting of CCR5.A. CHO CCR5 cells growing on coverslips were pre-treated (60 min, 4°C) with 100 nM chemokines and anti-CCR5 antibody 3A9 (green), then washed and incubated at 37°C for the indicated times. Cells were then acid washed to remove cell surface antibody, then fixed, permeabilized, and labeled for the ERC marker Rab11 (red) and DAPI (blue, nuclear staining) prior to analysis by confocal scanning microscopy are shown, scale bar = 20 μm. Throughout the experiment the ERC marker (red) remains in a discrete supranuclear spot that is visble in the center of the nuclei (blue) in the maximum intensity projections. Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to colocalize with the ERC marker. After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker. B. Individual Z-slice images from an identical experiment in which cells incubated with the indicated chemokines for 180 min were also labeled for either the ERC marker, Rab11 or the TGN marker, TGN38 (red). Slices in which the marked compartment is most abundant (through the middle of the nucleus for TGN, just above the nucleus for ERC) were chosen. While in cells treated with PSC-RANTES for 180 min, CCR5 colocalizes with TGN38 and does not colocalize with Rab11, in cells treated with 5P14-RANTES for 180 CCR5 colocalizes with Rab11 and does not colocalize with TGN38 scale bar = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414452&req=5

pone.0125396.g001: Ligand-directed post-endocytic sorting of CCR5.A. CHO CCR5 cells growing on coverslips were pre-treated (60 min, 4°C) with 100 nM chemokines and anti-CCR5 antibody 3A9 (green), then washed and incubated at 37°C for the indicated times. Cells were then acid washed to remove cell surface antibody, then fixed, permeabilized, and labeled for the ERC marker Rab11 (red) and DAPI (blue, nuclear staining) prior to analysis by confocal scanning microscopy are shown, scale bar = 20 μm. Throughout the experiment the ERC marker (red) remains in a discrete supranuclear spot that is visble in the center of the nuclei (blue) in the maximum intensity projections. Initially, CCR5 (green) is localized at the cell surface, but after 10 min incubation with either ligand it translocates to colocalize with the ERC marker. After 120 min incubation, CCR5 on cells treated with PSC-RANTES subsequently relocates from the ERC to accumulate in a perinuclear site (ring-shaped staining around the nuclei), while CCR5 in cells treated with 5P14-RANTES remains colocalized with the ERC marker. B. Individual Z-slice images from an identical experiment in which cells incubated with the indicated chemokines for 180 min were also labeled for either the ERC marker, Rab11 or the TGN marker, TGN38 (red). Slices in which the marked compartment is most abundant (through the middle of the nucleus for TGN, just above the nucleus for ERC) were chosen. While in cells treated with PSC-RANTES for 180 min, CCR5 colocalizes with TGN38 and does not colocalize with Rab11, in cells treated with 5P14-RANTES for 180 CCR5 colocalizes with Rab11 and does not colocalize with TGN38 scale bar = 20 μm.
Mentions: A full list of antibodies used in this study is provided in Table 1. For live microscopy, the anti-CCR5 monoclonal antibody Hek/1/85a (AB_369016, Serotec) was rhodamine-labeled using amine-reactive N-hydroxy-succinimidyl-Rhodamine (Pierce) according to the manufacturer’s instructions.

Bottom Line: The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors.This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV.Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Faculty of Medicine, University of Geneva. Geneva, Switzerland.

ABSTRACT
G protein-coupled receptors (GPCRs) are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling). The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

Show MeSH
Related in: MedlinePlus