Limits...
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.

Show MeSH
Spatial and temporal resolution of arrestin2-CCR5 association.A CHO-CCR5 cells stably transfected with arrestin2-GFP were treated with chemokine analogs (100 nM) as indicated and the redistribution of arrestin2-GFP was followed by live fluorescence microscopy. Images captured prior to (0 min) and after ligand treatment (6 min) are shown. B CHO-CCR5 cells stably transfected with arrestin2-GFP (green), preincubated with rhodamine-labeled anti-CCR5 antibody (red), were washed and then incubated 90 min at 37°C with chemokine analogs (100 nM) prior to image capture. Maximal intensity projections are shown. Scale bar = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414452&req=5

pone.0125396.g005: Spatial and temporal resolution of arrestin2-CCR5 association.A CHO-CCR5 cells stably transfected with arrestin2-GFP were treated with chemokine analogs (100 nM) as indicated and the redistribution of arrestin2-GFP was followed by live fluorescence microscopy. Images captured prior to (0 min) and after ligand treatment (6 min) are shown. B CHO-CCR5 cells stably transfected with arrestin2-GFP (green), preincubated with rhodamine-labeled anti-CCR5 antibody (red), were washed and then incubated 90 min at 37°C with chemokine analogs (100 nM) prior to image capture. Maximal intensity projections are shown. 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)

Spatial and temporal resolution of arrestin2-CCR5 association.A CHO-CCR5 cells stably transfected with arrestin2-GFP were treated with chemokine analogs (100 nM) as indicated and the redistribution of arrestin2-GFP was followed by live fluorescence microscopy. Images captured prior to (0 min) and after ligand treatment (6 min) are shown. B CHO-CCR5 cells stably transfected with arrestin2-GFP (green), preincubated with rhodamine-labeled anti-CCR5 antibody (red), were washed and then incubated 90 min at 37°C with chemokine analogs (100 nM) prior to image capture. Maximal intensity projections are shown. Scale bar = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414452&req=5

pone.0125396.g005: Spatial and temporal resolution of arrestin2-CCR5 association.A CHO-CCR5 cells stably transfected with arrestin2-GFP were treated with chemokine analogs (100 nM) as indicated and the redistribution of arrestin2-GFP was followed by live fluorescence microscopy. Images captured prior to (0 min) and after ligand treatment (6 min) are shown. B CHO-CCR5 cells stably transfected with arrestin2-GFP (green), preincubated with rhodamine-labeled anti-CCR5 antibody (red), were washed and then incubated 90 min at 37°C with chemokine analogs (100 nM) prior to image capture. Maximal intensity projections are shown. 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