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CHEMOKINES
From the School of Animal and Microbial Sciences,
University of Reading, United Kingdom, and the Department of
Pharmacology, The Medical School, Bristol, United Kingdom.
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.
(Blood. 2002;99:785-791) Chemokine receptor CCR5 is a member of the large
family of G-protein-coupled receptors (GPCRs) containing 7 membrane-spanning The mechanism by which the receptor number is regulated on the cell
surface, however, is unclear. Receptor number on the cell surface is a
balance between the rate of internalization and the rate of replacement
(recycling and new synthesis). There are 2 major routes whereby GPCRs
can be internalized after ligand binding. The first involves binding of
arrestin to the receptor, which results in a movement of the receptor
to clathrin-coated pits and internalization. The second pathway
involves caveolae and is independent of clathrin-coated pits. The
pathway dependent on clathrin-coated pits is still the best-known entry
system into cells11 and may be considered a default system
for degradation and recycling. Binding of arrestin-2 to the
phosphorylated receptor in turn initiates the internalization process
by binding to clathrin. Then the receptor-arrrestin-2 complex is
sequestered in clathrin-coated pits. By the action of dynamin, the
clathrin-coated pits are pinched off to become clathrin-coated
vesicles. Rab5- and rab7-dependent vesicle fusion processes are
involved in trafficking of the vesicles from early endosomes to late
endosomes to lysosomes.12-14
Caveolae are microdomains in the plasma membrane approximately 50 to
100 nm across. They are involved in several crucial cellular functions
such as endocytosis, photocytosis, transcytosis, calcium signaling, and
cholesterol transport. Biochemical studies have revealed the complex
molecular composition of caveolae.15 Caveolin, an integral
membrane protein (21-24 kd),16,17 and the distinct lipid
composition of the caveolae (enrichment of cholesterol, sphingolipids,
and glycolipids but the lack of phospholipids)18,19 are the
main molecular features of caveolae. Caveolae are capable of being
internalized in a regulated manner or under well-defined conditions.
Cholesterol is required for the maintenance of caveolae integrity and
function.20
Although the rate of internalization of a receptor is an important
factor in determining its level at the cell surface, the rate of
recycling and the rate of synthesis of new receptors are also
important. Some of the mechanisms of the recycling process are
understood. Internalized receptors are thought to have several potential fates. One fate is dephosphorylation of the receptor in
endosomes followed by recycling back to the plasma membrane. Sequentially, the receptors pass through late endosomes and the Golgi
and finally are transported back to the cell surface. Another fate is
that internalized receptors are degraded, which may result in receptor
down-regulation. Protein synthesis has not been shown to play a role in
receptor recycling.
Constituent mechanisms involved in these pathways can be inhibited
specifically by different chemicals. Formation of clathrin-coated pits
can be inhibited using 0.4 M sucrose or chlorpromazine,20 whereas the caveolae pathway is sensitive to filipin and nystatin treatment.20 Filipin flattens the caveolae, inhibits the
entry of cholera toxin, and releases several proteins of the cortical cytoskeleton, such as annexin II, alpha-actinin, ezrin, moesin, and
membrane-associated actin.21 Brefeldin A inhibits vesicle formation at the level of the Golgi apparatus, resulting in the disruption of the traffic between the Golgi and the endoplasmic reticulum,22 without impairing endosomal or lysosomal
function.23,24 Monensin blocks Golgi transport and
prevents the acidification of intracellular compartments and,
therefore, the recycling of receptors.25 Nocodazole
inhibits microtubule polymerization and, therefore, the transport of
endocytosed ligands from early to late endosomes.26
In this study we used these inhibitors to investigate the pathways that
are involved during CCR5 internalization. Work with the inhibitors has
been supported by confocal microscope studies of CCR5. We also
investigated which compartments in the cells are involved during the
recovery of the receptor and whether protein synthesis is involved.
Cells and materials
Chemicals
Internalization assay and flow cytometry analysis HeLa RC49, CHO.CCR5, and CHO.CCR5.CD4 cells were incubated with serum-free medium for 2 hours at 37°C harvested with 2 mM EDTA-phosphate-buffered saline (PBS) and then resuspended in medium without serum at 5 × 106 cells/mL. Cells were then incubated with chemokines (50 nM) for various times at 37°C, and washed in ice-cold PBS or PBS containing 1% FCS and 1% NaN3 for fluorescence-activated cell sorter (FACS) analysis. Cell surface-expressed CCR5 was detected by flow cytometry using anti-CCR5 antibody HEK/1/85a/7a and fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG. Cells were incubated for 1 hour at room temperature with HEK/1/85a/7a (saturating amounts of hybridoma supernatant), washed 3 times with PBS buffer containing 1% FCS and 1% NaN3, and incubated for 1 h with FITC-labeled anti-rat IgG. Samples were quantified on a FACScan, and data were analyzed with CellQuest software version 3.1 (Becton Dickinson, San Jose, CA). Relative CCR5 surface expression was calculated as 100× [mean channel of fluorescence (stimulated) mean channel of
fluorescence (negative control)/mean channel of fluorescence
(medium) mean channel of fluorescence (negative control)]
(%). CHO cells not expressing CCR5 and irrelevant monoclonal
antibodies were used for negative controls with similar results. Cell
surface expressed CD4 was detected in the same way, using the anti-CD4
monoclonal antibody ARP318 (5 µg per stain) and a corresponding
secondary FITC-labeled antibody.
Recycling of receptor Internalization was initiated as described. After 1-hour incubation with chemokines, the cells were washed 3 times in medium without FCS and resuspended in medium without FCS at 37°C. Samples were taken at different time points, and cells were washed in PBS buffer containing 1% FCS and 1% NaN3. Cells were stained with antibodies as described.Immunofluorescence Cells were grown on coverslips and incubated in medium without serum for 2 hours before treatment with chemokines for 1 hour. Cells were then washed with medium and incubated with the CCR5 antibody (HEK/1/85a/7a) for 1 hour at 37°C. After washing, the cells were incubated with the corresponding secondary FITC-labeled antibody for 1 hour, washed and fixed in ice-cold methanol, and mounted on glass slides. Images were taken using a Leica NT Confocal Imaging system. CHO.CCR5 or CHO.CCR5.CD4 cells were transiently transfected with pEGFP-arrestin-2 using LipofectAMINE (Invitrogen). Cells were treated with chemokines 48 hours after transfection and were stained with the CCR5 antibody and the corresponding secondary Rhodamine-labeled antibody.Data analysis Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). Statistical analysis was performed using Student t test (P < .05). Internalization data represent the means of at least 3 independent experiments.
Cell surface expression of CCR5 To investigate pathways of internalization of CCR5, we used 3 different cell lines: CHO cells that either expressed solely CCR5 (CHO.CCR5) or coexpressed CCR5 and CD4 (CHO.CCR5.CD4). We also used a HeLa cell line that expressed stably CCR5 and CD4.27 Cell surface expression of the receptor was determined using FACS analysis, and this showed comparable CCR5 expression levels of CHO.CCR5 and HeLa RC49, whereas the CHO.CCR5.CD4 cells expressed CCR5 at a much lower level (Figure 1A). CD4 expression levels on HeLa RC49 and CHO.CCR5.CD4 were similar (Figure 1B).
Inhibition of internalization Cells were pretreated with 0.4 M sucrose for 1 hour at 37°C in medium without FCS, and then an internalization assay was performed with 3 different chemokines (50 nM) for 1 hour. Sucrose treatment inhibited MIP-1 -induced internalization in all 3 cell lines tested. A significant inhibition of RANTES-induced and MIP-1 -induced internalization by sucrose was observed in CHO.CCR5.CD4 cells (Table
1). Treatment with 25 µg/mL
chlorpromazine for 1 hour gave results similar to those seen with
sucrose (data not shown).
We then pretreated the cells with nystatin (50 µg/mL) and filipin (5 µg/mL) for 1 hour and performed an internalization assay as described
(Table 2). Nystatin inhibited the
MIP-1
Effects of these inhibitors were also examined using confocal
microscopy (Figure 2). In control cells
CCR5 was localized to the plasma membrane, but after treatment with
chemokine CCR5 was seen to move to a vesicular compartment in the
cytosol. Sucrose, nystatin, and filipin prevented this movement in
CHO.CCR5 and CHO.CCR5.CD4 cells, in agreement with the data outlined
above. The role of arrestin-2 was examined by transient transfection of
pEGFP-arrestin-2 in CHO.CCR5 or CHO.CCR5.CD4 cells. This showed that
chemokine-induced activation of CCR5 leads to the redistribution of
arrestin-2 in cells, resulting in a colocalization of arrestin-2 and
CCR5 (Figure 2).
Effects of these inhibitors of membrane trafficking on the binding of
chemokines to the receptor and the function of the receptor was
assessed using a [35S]GTP Recovery of the receptor Recovery of the receptor to the cell surface was investigated after the internalization of CCR5 triggered by MIP-1 in CHO.CCR5 and
HeLa RC49 cells (Figure 3). After 1-hour
incubation with the chemokine at 37°C, cells were washed in medium
and incubated at 37°C for the times indicated. In some experiments,
protein synthesis in the cells was inhibited with cycloheximide (10 µg/mL) added during the recovery phase. After 120 minutes, most
receptors on the cell surfaces returned to control levels in both cell
systems used (Figure 3). In CHO.CCR5 cells we did not observe any
effects of cycloheximide on the recovery rate (Figure 3). It seemed
unlikely that protein synthesis was involved in receptor recovery. We
concluded that CCR5 recovery was solely caused by receptor
recycling.
Internalization is only one determinant of the number of the cell
surface receptors. Equally important is whether receptor recycling is
taking place simultaneously with internalization and whether this
recycling rate is influencing the internalization of the receptor. We
pretreated the cells with monensin and brefeldin A (Figure
4), which have been shown to
inhibit the function of the Golgi apparatus and, therefore, to inhibit
recycling. After pretreatment with monensin (50 µM), we observed the
inhibition of internalization but no effect on recycling. Monensin has
been shown to inhibit internalization by way of coated pits. Brefeldin A (5 µM) pretreatment of the cells had no significant effect on the
internalization rate.
These experiments did not allow determination of the recycling rate of
CCR5 receptor. Internalization was initiated as before, and the cells
were incubated for recovery, as described, in the presence or absence
of monensin and brefeldin A (Figure 5).
This experiment should allow normal internalization, and the chemicals used should only influence the recovery phase. Because we were unable
to observe an effect of monensin or brefeldin A on the recycling rate
of CCR5 (Figure 5), we concluded that recycling of CCR5 is independent
of the Golgi apparatus in the cells and that acidification of
intracellular compartments is not necessary for receptor
recycling.
These experiments showed that neither new synthesis of CCR5 nor
transport through the Golgi apparatus of the cell is involved in the
recycling phase. We treated the cells with nocodazole to investigate
earlier transport mechanisms in the cell. Treatment of cells with
nocodazole should inhibit transport of vesicles from early to late
endosomes. Pretreatment of cells with nocodazole at 37°C did not have
any effect on the recycling rate (Figure 6). Nevertheless, when the cells were
pretreated with nocodazole at 4°C, the recycling rate was diminished.
In the same experiment we found that internalization was inhibited, and
it seemed likely that incubation on ice disrupted the cell machinery
leading to a reduction in the recovery rate.
In this study we have examined the mechanisms that regulate the number of chemokine receptor CCR5 on cells and the effects of chemokines on these. We show that CCR5 internalization after chemokine treatment can occur through pathways involving clathrin-coated pits or caveolae. We also show that recycling of internalized CCR5 is independent of protein synthesis and depends on early endosomes. It has been well established that the chemokine receptor CCR5 acts as a
coreceptor for HIV-12 and that the levels of CCR5 on cells
influence the rate of entry of HIV-1. Hence, patients with reduced or
nonexistent levels of CCR5 on peripheral blood mononuclear cells
exhibit immunity against HIV-1 infection.7,8 In addition,
high expression of MIP-1 Two pathways have been described for the internalization of G-protein-coupled receptors, such as CCR5, after their activation. One pathway uses clathrin-coated pits.11 Activated receptor is phosphorylated and binds to arrestin proteins, and the complex is transported to clathrin-coated pits. Here it is internalized in vesicles and transported to endosomes where dephosphorylation takes place. The receptor is then recycled back to the cell surface. Some receptors are not recycled but may be transported from endosomes to lysosomes or proteasomes, and then they are degraded.12-14 A second pathway of internalization depends on caveolae. Caveolae are highly organized membrane structures that have been shown to be involved in the internalization of several GPCRs. To investigate which pathways are involved in CCR5 internalization and recycling, we used 3 different cell lines expressing CCR5. Two CHO cell lines expressing CCR5 alone (CHO.CCR5) or with CD4 (CHO.CCR5.CD4) have been used. CCR5 is expressed at a higher level in CHO.CCR5 cells than in the CHO.CCR5.CD4 cells. Additionally, we used a HeLa cell line (RC49) that expresses CCR5 and CD4; levels of CCR5 were comparable to those in CHO.CCR5 cells. First, we investigated the role of the 2 pathways of internalization
using selective inhibitors, and similar results were seen in each of
the 3 cell lines. Internalization of CCR5 from MIP-1 CCR5 was also affected by inhibitors of caveolae-dependent
internalization pathways. In CHO.CCR5 cells, treatment with nystatin (50 µg/mL) inhibited MIP-1 Next, we investigated recovery mechanisms for CCR5 after
internalization. Recovery experiments in CHO.CCR5 and HeLa RC49 cells showed that after 120 minutes' incubation, the level of receptor on
the cell surface was back to nearly 100%. In CHO.CCR5 cells, we could
not observe any effect of cycloheximide treatment on the recovery rate,
so protein synthesis was unlikely to be involved in CCR5 recovery in
these cells. Receptor recycling is thought to use several steps in the
cell machinery. Receptors are transported to early and late endosomes,
where dephosphorylation and resensitization takes place before
recycling. To examine these processes, we used monensin treatment to
inhibit the acidification of intracellular compartments and brefeldin A
to block the translocation of protein from the endoplasmic reticulum to
the Golgi. After pretreatment of the cells with these compounds,
however, we could not observe any MIP-1 The experimental protocol was changed to circumvent these problems. Chemokine-induced internalization was performed in cells in the absence of inhibitors, and during the recovery phase brefeldin A or monensin was added to the cells. Using this experimental protocol, we did not observe any significant influence of monensin or brefeldin A on the recovery rate of CCR5. It seems likely that CCR5 is recycled back to the cell surface without passing through the Golgi apparatus in the cells. Nocodazole has been described as an inhibitor of the transport of vesicles between early and late endosomes. We tested the effects of this substance on CCR5 recycling by pretreating cells. There was no effect of nocodazole on CCR5 recovery after internalization. It seems unlikely that late endosomes are involved in the recovery of the receptor. While this work was nearing completion, a study was published30 that showed that CCR5 was internalized to endosomal vesicles with properties similar to those described for recycling endosomes. These data, obtained with different techniques, are in full agreement with those in the current study. Based on the current observations, the mechanism of CCR5 internalization and recycling seems to involve clathrin-coated pits and caveolae. After internalization, CCR5 is transported to early endosomes and recycled back to the cell surface. There is no evidence for the involvement of late endosomes, Golgi apparatus, or protein synthesis.
We thank Dr Jane McKeating for her involvement in the inception of the project and for generating cell lines and antibodies. We thank Dr Christine Shotton for monoclonal antibodies to CCR5 and Dr Lloyd Czaplewski (British Biotech) for various chemokine reagents. We thank the Centralized Facility for AIDS Reagents, supported by EU Programme EVA (contract BMH4 97/2515) and the United Kingdom Medical Research Council. We also thank Dr David Kabat for the HeLa RC49 cell line.
Submitted June 13, 2001; accepted September 16, 2001.
Supported by a project grant from the Biotechnology and Biological Sciences Research Council.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Philip G. Strange, School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, RG6 6AJ, United Kingdom; e-mail: p.g.strange{at}rdg.ac.uk.
1.
Blanpain C, Migeotte I, Lee B, et al.
CCR5 binds multiple CC-chemokines: MCP-3 acts as a natural antagonist.
Blood.
1999;94:1899-1905
2.
Alkhatib G, Combadiere C, Broder CC, et al.
CC CKR5: a RANTES, MIP-1 3. Choe H, Farzan M, Sun Y, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996;85:1135-1148[CrossRef][Medline] [Order article via Infotrieve]. 4. Doranz BJ, Rucker J, Yi Y, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell. 1996;85:1149-1158[CrossRef][Medline] [Order article via Infotrieve]. 5. Dragic T, Litwin V, Allaway GP, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature. 1996;381:667-673[CrossRef][Medline] [Order article via Infotrieve]. 6. Littman DR. Chemokine receptors: keys to AIDS pathogenesis? Cell. 1998;93:677-680[CrossRef][Medline] [Order article via Infotrieve]. 7. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene: Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study Vol 273. Multicenter Hemophilia Cohort Study: San Francisco City Cohort, ALIVE Study. Science.; 1996:1856-1862. 8. Michael NL, Louie LG, Rohrbaugh AL, et al. The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression. Nat Med. 1997;10:1160-1162.
9.
Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P.
Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.
Science.
1995;270:1811-1815 10. Cocchi F, DeVico AL, Yarchoan R, et al. Higher macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels from CD8+ T cells are associated with asymptomatic HIV-1 infection. Proc Natl Acad Sci U S A. 2000;25:13812-13817.
11.
Orlandi PA, Fishman PH.
Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains.
J Cell Biol.
1998;141:905-915 12. Novick P, Zerial M. The diversity of Rab proteins in vesicle transport. Curr Opin Cell Biol. 1997;9:496-504[CrossRef][Medline] [Order article via Infotrieve].
13.
Barak LS, Tiberi M, Freedman NJ, Kwatra MM, Lefkowitz RJ, Caron MG.
A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration.
J Biol Chem.
1994;269:2790-2795 14. Campbell PT, Hnatowich M, O'Dowd BF, Caron MG, Lefkowitz RJ, Hausdorff WP. Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. Mol Pharmacol. 1991;39:192-198[Abstract]. 15. Anderson RG. The caveolae membrane system. Annu Rev Biochem. 1998;67:199-225[CrossRef][Medline] [Order article via Infotrieve]. 16. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. Caveolin, a protein component of caveolae membrane coats. Cell. 1992;68:673-682[CrossRef][Medline] [Order article via Infotrieve].
17.
Kurzchalia TV, Dupree P, Parton RG, et al.
VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.
J Cell Biol.
1992;118:1003-1014 18. Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992;68:533-544[CrossRef][Medline] [Order article via Infotrieve]. 19. Fiedler K, Kobayashi T, Kurzchalia TV, Simons K. Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry. 1993;32:6365-6373[CrossRef][Medline] [Order article via Infotrieve].
20.
Okamoto Y, Ninomiya H, Miwa S, Masaki T.
Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.
J Biol Chem.
2000;275:6439-6446 21. Harder T, Kellner R, Parton RG, Gruenberg J. Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol. Mol Biol Cell. 1997;3:533-545.
22.
Klausner RD, Donaldson JG, Lippincott-Schwartz J.
Brefeldin A: insights into the control of membrane traffic and organelle structure.
J Cell Biol.
1992;116:1071-1080
23.
Wood SA, Brown WJ.
The morphology but not the function of endosomes and lysosomes is altered by brefeldin A.
J Cell Biol.
1992;119:273-285
24.
Strous GJ, van Kerkhof P, van Meer G, Rijnboutt S, Stoorvogel W.
Differential effects of brefeldin A on transport of secretory and lysosomal proteins.
J Biol Chem.
1993;268:2341-2347
25.
Law P, Erickson LJ, El-Kouhen R, et al.
Receptor density and recycling affect the rate of agonist desensitization of µ-opioid receptor.
Mol Pharmacol.
2000;58:388-398 26. Gruenberg J, Maxfield FR. Membrane transport in the endocytic pathway. Curr Opin Cell Biol. 1995;4:552-563.
27.
Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D.
Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1.
J Virol.
1998;72:2855-2864 28. Mundell SJ, Matharu AL, Kelly E, Benovic JL. Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking. Biochemistry. 2000;42:12828-12836.
29.
Gardner BR, Hall DA, Strange PG.
Pharmacological analysis of dopamine stimulation of [35S]GTP
30.
Signoret N, Pelchen-Matthews A, Mack M, Proudfoot AE, Marsh M.
Endocytosis and recycling of the HIV coreceptor CCR5.
J Cell Biol.
2000;151:1281-1294
© 2002 by The American Society of Hematology.
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|
|
|
|
 |
|
 H. Yuling, X. Ruijing, J. Xiang, J. Yanping, C. Lang, L. Li, Y. Dingping, T. Xinti, L. Jingyi, T. Zhiqing, et al. CD19+CD5+ B Cells in Primary IgA Nephropathy J. Am. Soc. Nephrol., November 1, 2008; 19(11): 2130 - 2139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
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|
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|
|
 |
|
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|
|
|
|
|
|
R. Bonecchi, E. M. Borroni, A. Anselmo, A. Doni, B. Savino, M. Mirolo, M. Fabbri, V. R. Jala, B. Haribabu, A. Mantovani, et al. Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation Blood, August 1, 2008; 112(3): 493 - 503. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
A. Chakera, R. M. Seeber, A. E. John, K. A. Eidne, and D. R. Greaves The Duffy Antigen/Receptor for Chemokines Exists in an Oligomeric Form in Living Cells and Functionally Antagonizes CCR5 Signaling through Hetero-Oligomerization Mol. Pharmacol., May 1, 2008; 73(5): 1362 - 1370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Delhaye, A. Gravot, D. Ayinde, F. Niedergang, M. Alizon, and A. Brelot Identification of a Postendocytic Sorting Sequence in CCR5 Mol. Pharmacol., December 1, 2007; 72(6): 1497 - 1507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
Y. Saita, E. Kodama, M. Orita, M. Kondo, T. Miyazaki, K. Sudo, K. Kajiwara, M. Matsuoka, and Y. Shimizu Structural Basis for the Interaction of CCR5 with a Small Molecule, Functionally Selective CCR5 Agonist. J. Immunol., September 1, 2006; 177(5): 3116 - 3122. [Abstract] [Full Text] [PDF]
|
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C. Otero, M. Groettrup, and D. F. Legler Opposite Fate of Endocytosed CCR7 and Its Ligands: Recycling versus Degradation J. Immunol., August 15, 2006; 177(4): 2314 - 2323. [Abstract] [Full Text] [PDF]
|
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|
 N. Michel, K. Ganter, S. Venzke, J. Bitzegeio, O. T. Fackler, and O. T. Keppler The Nef Protein of Human Immunodeficiency Virus Is a Broad-Spectrum Modulator of Chemokine Receptor Cell Surface Levels That Acts Independently of Classical Motifs for Receptor Endocytosis and G{alpha}i Signaling Mol. Biol. Cell, August 1, 2006; 17(8): 3578 - 3590. [Abstract] [Full Text] [PDF]
|
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|
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|
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R. Schiavo, D. Baatar, P. Olkhanud, F. E. Indig, N. Restifo, D. Taub, and A. Biragyn Chemokine receptor targeting efficiently directs antigens to MHC class I pathways and elicits antigen-specific CD8+ T-cell responses Blood, June 15, 2006; 107(12): 4597 - 4605. [Abstract] [Full Text] [PDF]
|
|
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|
|
|
C. Pastori, B. Weiser, C. Barassi, C. Uberti-Foppa, S. Ghezzi, R. Longhi, G. Calori, H. Burger, K. Kemal, G. Poli, et al. Long-lasting CCR5 internalization by antibodies in a subset of long-term nonprogressors: a possible protective effect against disease progression Blood, June 15, 2006; 107(12): 4825 - 4833. [Abstract] [Full Text] [PDF]
|
|
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|
 T. L. Thirkill, H. Vedagiri, and G. C. Douglas Macaque Trophoblast Migration toward RANTES Is Inhibited by Cigarette Smoke-Conditioned Medium Toxicol. Sci., June 1, 2006; 91(2): 557 - 567. [Abstract] [Full Text] [PDF]
|
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M. S. K. Sutherland, R. J. Sanderson, K. A. Gordon, J. Andreyka, C. G. Cerveny, C. Yu, T. S. Lewis, D. L. Meyer, R. F. Zabinski, S. O. Doronina, et al. Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates J. Biol. Chem., April 14, 2006; 281(15): 10540 - 10547. [Abstract] [Full Text] [PDF]
|
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J. E. Christensen, C. de Lemos, T. Moos, J. P. Christensen, and A. R. Thomsen CXCL10 Is the Key Ligand for CXCR3 on CD8+ Effector T Cells Involved in Immune Surveillance of the Lymphocytic Choriomeningitis Virus-Infected Central Nervous System J. Immunol., April 1, 2006; 176(7): 4235 - 4243. [Abstract] [Full Text] [PDF]
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 F. F. Hamdan, M. Audet, P. Garneau, J. Pelletier, and M. Bouvier High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based {beta}-Arrestin2 Recruitment Assay J Biomol Screen, August 1, 2005; 10(5): 463 - 475. [Abstract] [PDF]
|
|
|
|
|
|
C. Barassi, E. Soprana, C. Pastori, R. Longhi, E. Buratti, F. Lillo, C. Marenzi, A. Lazzarin, A. G. Siccardi, and L. Lopalco Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy J. Virol., June 1, 2005; 79(11): 6848 - 6858. [Abstract] [Full Text] [PDF]
|
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N. Signoret, L. Hewlett, S. Wavre, A. Pelchen-Matthews, M. Oppermann, and M. Marsh Agonist-induced Endocytosis of CC Chemokine Receptor 5 Is Clathrin Dependent Mol. Biol. Cell, February 1, 2005; 16(2): 902 - 917. [Abstract] [Full Text] [PDF]
|
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A. Biragyn, P. A. Ruffini, M. Coscia, L. K. Harvey, S. S. Neelapu, S. Baskar, J.-M. Wang, and L. W. Kwak Chemokine receptor-mediated delivery directs self-tumor antigen efficiently into the class II processing pathway in vitro and induces protective immunity in vivo Blood, October 1, 2004; 104(7): 1961 - 1969. [Abstract] [Full Text] [PDF]
|
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 |
|
 H. A. W. Tawfeek and A. B. Abou-Samra Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E704 - E710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-H. Fan, L. A. Lapierre, J. R. Goldenring, J. Sai, and A. Richmond Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis Mol. Biol. Cell, May 1, 2004; 15(5): 2456 - 2469. [Abstract] [Full Text] [PDF]
|
|
|
|
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V. Ramakrishna, J. F. Treml, L. Vitale, J. E. Connolly, T. O'Neill, P. A. Smith, C. L. Jones, L.-Z. He, J. Goldstein, P. K. Wallace, et al. Mannose Receptor Targeting of Tumor Antigen pmel17 to Human Dendritic Cells Directs Anti-Melanoma T Cell Responses via Multiple HLA Molecules J. Immunol., March 1, 2004; 172(5): 2845 - 2852. [Abstract] [Full Text] [PDF]
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 J. P. Camina, M. C. Carreira, S. El Messari, C. Llorens-Cortes, R. G. Smith, and F. F. Casanueva Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a Endocrinology, February 1, 2004; 145(2): 930 - 940. [Abstract] [Full Text] [PDF]
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 Z. Qiuping, L. Qun, H. Chunsong, Z. Xiaolian, H. Baojun, Y. Mingzhen, L. Chengming, H. Jinshen, G. Qingping, Z. Kejian, et al. Selectively Increased Expression and Functions of Chemokine Receptor CCR9 on CD4+ T Cells from Patients with T-Cell Lineage Acute Lymphocytic Leukemia Cancer Res., October 1, 2003; 63(19): 6469 - 6477. [Abstract] [Full Text] [PDF]
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S. Venkatesan, J. J. Rose, R. Lodge, P. M. Murphy, and J. F. Foley Distinct Mechanisms of Agonist-induced Endocytosis for Human Chemokine Receptors CCR5 and CXCR4 Mol. Biol. Cell, August 1, 2003; 14(8): 3305 - 3324. [Abstract] [Full Text] [PDF]
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F. Huttenrauch, A. Nitzki, F.-T. Lin, S. Honing, and M. Oppermann beta -Arrestin Binding to CC Chemokine Receptor 5 Requires Multiple C-terminal Receptor Phosphorylation Sites and Involves a Conserved Asp-Arg-Tyr Sequence Motif J. Biol. Chem., August 16, 2002; 277(34): 30769 - 30777. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
S binding
assay.
, control; 
, 75 minutes;
, 90 minutes; 
, 120 minutes. Data represent means ± SEM of
at least 3 independent experiments. **P < .01;
*P < .05, relative to control (MIP-1
