|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-07-1965.
CHEMOKINES
From the Department of Veterans Affairs, Nashville, and
the Departments of Cancer Biology and Surgery, Vanderbilt
University School of Medicine, Nashville, TN.
Intracellular trafficking of chemokine receptors plays an important
role in fine-tuning the functional responses of neutrophils and
lymphocytes in the inflammatory process and HIV infection. Although
many chemokine receptors internalize through clathrin-coated pits, regulation of the receptor trafficking is not fully
understood. The present study demonstrated that CXCR2 was colocalized
with transferrin and low-density lipoprotein (LDL) after agonist
treatment for different periods of time, suggesting 2 intracellular
trafficking pathways for this receptor. CXCR2 was colocalized with Rab5
and Rab11a, which are localized in early and recycling endosomes, respectively, in response to agonist stimulation for a short period of
time, suggesting a recycling pathway for the receptor trafficking. However, overexpression of a dominant-negative Rab5-S34N mutant significantly attenuated CXCR2 sequestration. The internalized CXCR2
was recycled back to the cell surface after removal of the agonist and
recovery of the cells, but receptor recycling was inhibited by
overexpression of a dominant-negative Rab11a-S25N mutant. After
prolonged (4-hour) agonist treatment, CXCR2 exhibited significantly
increased colocalization with Rab7, which is localized in late
endosomes. The colocalization of CXCR2 with LDL and LAMP-1 suggests
that CXCR2 is targeted to lysosomes for degradation after prolonged
ligand treatment. However, the colocalization of CXCR2 with Lamp1 was
blocked by the overexpression of a dominant-negative Rab7-T22N mutant.
In cells overexpressing Rab7-T22N, CXCR2 was retained in the Rab5- and
Rab11a-positive endosomes after prolonged (4-hour) agonist treatment.
Our data suggest that the intracellular trafficking of CXCR2 is
differentially regulated by Rab proteins.
(Blood. 2003;101:2115-2124) Chemokines are a family of small proteins that
regulate leukocyte trafficking and are therefore important in lymphoid
organogenesis, inflammation, and host defense. Based on the presence of
the conserved cysteines near the N-terminus of chemokines, chemokines
can be classified into CC, CXC, CX3C, and C
subfamilies.1-4 The effects of chemokines on target cells
are mediated by a family of G-protein-coupled 7-transmembrane
receptors (GPCRs). Chemokine receptors are expressed predominantly in
neutrophils and lymphocytes. To date, 19 chemokine receptors have been
identified, including 11 CC chemokine receptors (CCR1-11), 6 CXC
chemokine receptors (CXCR1-6), and one for each C and CX3C chemokine
receptor.4 These receptors play important roles in
inflammatory response, chemotaxis, immune cell development, leukocyte
homing, and angiogenesis.5,6 Some of the chemokine receptors (eg, CCR5 and CXCR4) act as coreceptors in HIV
infection.7
Agonist occupancy of chemokine receptors, like other types of GPCRs,
activates the coupled heterotrimeric G proteins that mobilize
intracellular second messengers such as inositol triphosphates, cyclic
adenosine monophosphate, and intracellular free calcium.8 The receptors are rapidly phosphorylated by G-protein-coupled receptor
kinases or protein kinase C, followed by receptor desensitization and
internalization.9-11 Many chemokine receptors
internalize through clathrin-coated pits, a process facilitated by
adaptor proteins such as Intracellular trafficking of chemokine receptors plays an important
role in regulation of the functional responses of the chemokine
receptor expressing cells in inflammatory processes and HIV infection.
Increasing lines of evidence show the critical role of receptor
internalization in receptor-mediated chemotaxis. Studies on CXCR2, a
major chemokine receptor expressed in neutrophils, have demonstrated
that blocking the receptor internalization reduces receptor-mediated
chemotaxis.12,13 The responses of neutrophils, the major
infiltrate in the course of acute inflammation, could be regulated in
part by the processes involving chemokine receptor internalization.18-20 Moreover, chemokine-mediated block
to HIV entry is associated with CCR5 internalization
efficiency.21 However, the mechanisms underlying
intracellular trafficking of chemokine receptors are not fully understood.
Intracellular trafficking of membrane receptors is tightly regulated by
a subfamily of ras-like small GTPases (Rab GTPases). Approximately 40 members of Rab GTPases have been identified, and each is believed to be
specifically associated with a particular organelle or pathway. Among
them, Rab4 is considered to be localized in early and recycling
endosomes.22 Rab5 and Rab15 are localized in early
endosomes,22-25 Rab11 and Rab25 are localized in recycling endosomes,23,26-28 and Rab7 and Rab9 are localized in late
endosomes.29,30 Because Rab9 is also present in the
trans-Golgi network and regulates transport from late endosomes to the
trans-Golgi network,30 the more likely candidate
responsible for the continuous fusion events between late endosomes and
lysosomes is Rab7. Although the potential role of these Rab GTPases in
the trafficking of GPCRs is largely unknown, some GPCRs, such as the
dopamine D2 receptor and the Plasmids
Cell culture and transfection
Confocal visualization of agonist-induced internalization of CXCR2 Confocal microscopy was performed on a Zeiss LSM-410 laser scanning microscope using a Zeiss 63 × 1.3 numerical aperture (NA) oil immersion lens. HEK293 cells coexpressing CXCR2 and EGFP-Rab proteins were grown on coverslips. Cells were treated with carrier buffer or CXCL8 for different times and were fixed with methanol. Cells were washed with phosphate-buffered saline (PBS) and were incubated with a mouse monoclonal CXCR2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes. Cells were washed and incubated with a rhodamine-conjugated antimouse antibody (Molecular Probes) for 30 minutes. Confocal microscopy was performed using dual excitation (488 nm, EGFP; 568 nm, rhodamine or Cy3) and emission (515-540 nm, EGFP; 590-610 nm, rhodamine or Cy3) filter sets. To study the colocalization of CXCR2 with Lamp-1, cells were treated with CXCL8 for 4 hours, fixed in methanol. Cells were incubated with an antibody mixture containing a rabbit CXCR2 antibody (Santa Cruz Biotechnology) and a mouse monoclonal Lamp-1 antibody (PharMingen, San Jose, CA) for 30 minutes. Cells were washed and incubated with an antibody mixture containing a rhodamine-conjugated antirabbit antibody (Molecular Probes, Eugene, OR) and a fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Molecular Probes) for 30 minutes. Confocal microscopy was performed using dual-excitation (488 nm, FITC; 568 nm, rhodamine) and emission (515-540 nm, FITC; 590-610 nm rhodamine) filter sets. Specificity of labeling and absence of signal crossover were established by examination of single-labeled samples. To determine the colocalization of CXCR2 with transferrin or LDL, HEK293 cells stably expressing CXCR2 were incubated with transferrin-Texas Red (25 µg/mL) (Molecular Probes) or with Dil-LDL (40 µg/mL) (Molecular Probes) for 1 hour or 4 hours at 37°C in the presence of 200 nM CXCL8. Cells were washed with ice-cold serum-free medium, fixed in methanol, and processed for further immunostaining of CXCR2. Cells were incubated with a monoclonal CXCR2 antibody for 30 minutes followed by incubation with an FITC-conjugated antimouse antibody for 30 minutes. Incubation with antibodies was performed at room temperature. Colocalization of CXCR2 with transferrin or LDL was visualized by confocal microscopy. The percentage of colocalized CXCR2 was analyzed by calculating the colocalized receptor fluorescence (yellow) from the total internalized receptor fluorescence (yellow and green).Ligand-receptor complex internalization assay The acid-wash technique12 was used to determine the kinetics of CXCL8-induced internalization of CXCR2. HEK293 cells expressing CXCR2 were grown to confluence on 24-well plates precoated with 0.1 mg/mL poly-L-lysine (Sigma; MWt 30 000-70 000) for 1 hour and washed once with distilled water before use. Cells were incubated at 4°C in 0.5 mL serum-free DMEM containing 75 nCi/mL (2.775 KBq/mL) 125I-CXCL8 for 1 hour. The medium was subsequently removed; 1 mL ice-cold serum-free DMEM was added carefully to each well and aspirated, and then another 1-mL aliquot of ice-cold serum-free DMEM was added before incubation at 37°C for the indicated time. The medium was removed, and the cells were incubated with 1 mL ice-cold 0.2 M acetic acid with 0.5 M NaCl for 6 minutes. After incubation, the cells were washed once with 1-mL ice-cold serum-free DMEM and were lysed with 1 mL 1% sodium dodecyl sulfate (SDS) with 0.1 N NaOH (lysis solution). The radioactive cell lysate was then counted on a -counter (Gamma 5500; Beckman,
Muskegon, MI). Total cell surface receptor binding was measured after
incubation with 125I-CXCL8 medium followed by washing the
cells with ice-cold serum-free DMEM. Nonspecific binding was measured
by adding 200 mM acetic acid with 0.5 M NaCl after incubation with
125I-CXCL8 in binding medium. Calculation of the
percentage of internalized receptor was performed as described
before.12
Fluorescence-activated cell sorting analysis HEK293 cells expressing CXCR2 and Rab11a proteins were incubated in 20 mM HEPES (N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered DMEM at 37°C for 30 minutes in the presence or absence of CXCL8 (100 nM). Cells were then washed in ice-cold medium and incubated in medium at 37°C for different times as indicated. Cells were incubated with a monoclonal CXCR2 antibody (Santa Cruz Biotechnology) at 4°C for 1 hour, then washed with ice-cold medium followed by incubation with FITC-conjugated antimouse immunoglobulin G (IgG) at 4°C for 30 minutes. Cells were washed and fixed in 2% formaldehyde in PBS and were analyzed in FACScan equipped with CellQuest software (Becton Dickinson, Franklin Lakes, NJ).
Time-dependent internalization of CXCR2 into different endosomal compartments It has been postulated that CXCR2 is internalized into endosomal compartments in response to agonist stimulation.13 The small GTPases Rab5, Rab11a, and Rab7 are localized in early, recycling, and late endosomes, respectively, and they broadly regulate the intracellular trafficking and membrane fusion of endocytic vesicles.37 Here we determined whether agonist induced the internalization of CXCR2 into these small GTPase-containing endosomes. To visualize the colocalization of CXCR2 with these EGFP-Rab proteins, HEK293 cells and RBL-2H3 cells stably expressing CXCR2 were transiently transfected with plasmids encoding EGFP-tagged Rab5, Rab11a, and Rab7, respectively. HEK293 cells were treated with CXCL8 (200 nM) for different times, and the localization of CXCR2 and EGFP-Rab proteins was determined by confocal microscopy. As shown in Figures 1-3, before agonist stimulation, CXCR2 was expressed predominantly on the cell surface, whereas EGFP-tagged Rab5, Rab11a, and Rab7 were located largely in the cytoplasm (Figures 1A,2A,3A). In response to agonist stimulation, the internalized CXCR2 was found to be partially colocalized with EGFP-Rab5 (Figure 1B-C) and EGFP-Rab11a (Figure 2B-C), and colocalization lasted for at least 1 hour (Figures 1C,2C). After prolonged (4-hour) agonist exposure, CXCR2 was retained in the cytoplasm, but colocalization of the internalized receptor with EGFP-Rab5 and EGFP-Rab11a was significantly decreased (Figures 1D,2D). These data suggest that CXCR2 might have been transported to late endosomes after prolonged agonist stimulation. This is supported by the observation that after agonist treatment for 1 hour, little colocalization of CXCR2 with EGFP-Rab7 was observed (Figure 3B), whereas after treatment for 4 hours and 6 hours, there was significant colocalization of CXCR2 with EGFP-Rab7 (Figure 3C-D). Similarly, in RBL-2H3 cells coexpressing CXCR2 and the EGFP-tagged Rab proteins, CXCL8 treatment for 1 hour resulted in significant colocalization of the receptor with EGFP-Rab5 (Figure 1E) and EGFP-Rab11a (Figure 2E) but little colocalization with EGFP-Rab7 (Figure 3E). After 4 hours of agonist treatment, colocalization of the receptor with EGFP-Rab5 (Figure 1F) and EGFP-Rab11a (Figure 2F) was remarkably reduced. Instead, a significant increase was exhibited in the colocalization of the receptor with EGFP-Rab7 (Figure 3F). These data suggest that internalized CXCR2 was transported to Rab7-positive late endosomes during this time.
It may raise questions about whether the CXCR2 antibody used
specifically recognizes the receptor and whether the overexpressed EGFP-Rab proteins exhibit the same pattern and behavior as the endogenous ones. The specificity of CXCR2 immunostaining was determined in HEK293 cells stably expressing or not expressing CXCR2. Only the
cells expressing CXCR2 exhibited specific fluorescence on the cell
surface (Figure 4A), whereas the cells
without transfection of the receptor cDNA did not show any specific
fluorescence (Figure 4B). To determine whether CXCR2 colocalizes with
the native Rab proteins, HEK293 cells stably expressing CXCR2 were
treated with CXCL8 for 1 hour or 4 hours, followed by immunostaining
with an antibody mixture containing a monoclonal CXCR2 antibody and a rabbit antibody for Rab5, Rab11a, or Rab7. As shown in Figure 4, CXCR2
exhibited partial colocalization with Rab5 (Figure 4C-D) and Rab11a
(Figure 4E-F) after 1 hour of agonist treatment. After 4 hours of
agonist treatment, CXCR2 exhibited partial colocalization with native
Rab7 (Figure 4G-H). These results, taken together with the data shown
in Figures 1 to 3, clearly demonstrate that the overexpressed EGFP-Rab
proteins exhibited the same behavior as the endogenous Rab proteins,
consistent with the results obtained previously.35
Time-dependent colocalization of CXCR2 with Rab5, Rab11a, and Rab7
strongly suggests that there are at least 2 trafficking pathways for
the receptor. At the early stage of trafficking, the internalized
receptors are delivered predominantly to the early and recycling
endosomes. However, after prolonged agonist treatment, the receptors
are transported to late endosomes/lysosomes. By analyzing the
percentage of CXCR2 colocalized with transferrin, which is known to be
transported quickly into early and recycling endosomes,38
it was revealed that 84.9% ± 9.2% of internalized receptors were
colocalized with transferrin in response to agonist treatment for 1 hour (Figure 5A,E). Colocalization was
decreased to 30.2% ± 6.4% after agonist treatment for 4 hours
(Figure 5B,E) (P < .01). Using LDL as a lysosomal
probe39 to analyze the percentage of CXCR2 transported to
lysosomes, it was revealed that 18.8% ± 6.4% of internalized
receptors were colocalized with LDL after agonist treatment for 1 hour
(Figure 5C,E), but colocalization was remarkably increased to
67.8% ± 8.5% after 4 hours of agonist exposure (Figure 5D-E)
(P < .01).
CXCR2 internalization is regulated by Rab5 To assess whether these Rab GTPases affect the receptor endocytosis, HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids for EGFP-tagged Rab5, Rab11a, and Rab7, respectively, and ligand-bound receptor internalization was measured by a radioligand binding assay. Ligand-bound CXCR2 internalization was not affected by the overexpression of EGFP-tagged wild-type Rab5, Rab11a, or Rab7 (Figure 6A-C). Interestingly, overexpression of a dominant-negative mutant Rab5-S34N significantly attenuated the internalization of CXCR2 (Figure 6A), whereas the dominant-negative mutants of Rab11a and Rab7 exhibited little effect on receptor internalization (Figure 6B-C). The inhibitory effect of the dominant-negative Rab5 mutant on the internalization of CXCR2 was confirmed by confocal microscopy. As shown in Figure 7, CXCR2 was internalized into cells expressing EGFP-Rab5-Q79L (constitutively active mutant) in response to agonist treatment for 30 minutes. The internalized receptor was colocalized with the EGFP-Rab5-Q79L (Figure 7A-B). However, the dominant-negative EGFP-Rab5-S34N mutant exhibited a diffuse cytosolic distribution, in contrast to the wild-type EGFP-Rab5 protein. Agonist-activated CXCR2 did not colocalize with the EGFP-Rab5-S34N mutant in this compartment (Figure 7C-D). Although some CXCR2 immunostaining was observed inside the cells expressing EGFP-Rab5-S34N, most CXCR2 staining was localized to vesicles that appeared to be associated with or to be retained close to the cell surface (Figure 7C). These data suggest the regulation of CXCR2 internalization by Rab5.
CXCR2 recycling is regulated by Rab11a Previous studies on neutrophils have shown that internalized CXCR2 is recycled back to the plasma membrane on free-ligand (CXCL8) removal and recovery of the cells at 37°C.40 Rab11a, which is localized in the recycling endosomes, has been demonstrated to play a role in receptor recycling. Colocalization of the agonist-activated CXCR2 with EGFP-Rab11a (Figure 2) suggests that Rab11a regulates CXCR2 recycling. To test this hypothesis, we analyzed by fluorescence-activated cell sorter (FACS) the kinetics of recycling of CXCR2 on CXCL8 removal and recovery of the cells after CXCL8 exposure for 30 minutes at 37°C. Receptor re-expression levels following ligand removal were compared with the levels of their expression in control cells in which CXCL8-induced CXCR2 internalization was not elicited. As shown in Figure 8, without recovery, the internalized receptor levels are comparable among control cells (transfected with vector; Figure 8A,J), cells overexpressing Rab11a (Figure 8D,J), and cells overexpressing Rab11a-S25N (Figure 8G,J). Following removal of the agonist and recovery of the cells at 37°C, CXCR2 was re-expressed on the cell membrane. On incubation at 37°C for 30 minutes, CXCR2 expression was restored to 55.4% ± 4.9% of the level in the control cells (Figure 8B,J). Recovery of receptor expression to 76.9% ± 6.3% was observed after 1 hour of incubation (Figure 8C,J). Overexpression of wild-type Rab11a did not affect the recovery of CXCR2 (57% ± 6.3% for 30 minutes of incubation, 78.4% ± 8.1% for 1 hour of incubation; Figure 8E,F,J). In contrast, in cells overexpressing Rab11a-S25N, the recovery of CXCR2 levels was significantly reduced (35.5% ± 2.4% for 30 minutes of incubation, 44.8% ± 3.8% for 1 hour of incubation; Figure 8H-J).
The effect of Rab11a on the recycling of CXCR2 was confirmed by
confocal microscopy. HEK293 cells were cotransfected with plasmids for
CXCR2, together with EGFP-Rab11a-S20V (constitutively active mutant) or
EGFP-Rab11a-S25N (dominant-negative mutant). Figure
9 shows the different pattern of
distribution between EGFP-Rab11a-S20V and EGFP-Rab11a-S25N.
EGFP-Rab11a-S20V was punctated in the cytoplasm as the expression
pattern of wild-type Rab11a (Figure 9B,D). In contrast,
EGFP-Rab11a-S25N exhibited diffuse cytosolic distribution (Figure
9F,H). To observe the receptor recycling, we incubated the
cells with CXCL8 for 30 minutes at 37°C and removed the agonist by
washing the cells with agonist-free medium. Cells either were fixed
immediately or were recovered by incubation in agonist-free medium for
1 hour at 37°C. As shown in Figure 9, without recovery, CXCR2 was
localized in the cytoplasm in the cells expressing either EGFP-Rab11a-S20V (Figure 9A-B) or EGFP-Rab11a-S25N (Figure 9E,F). Following removal of the agonist and recovery of the cells at 37°C
for 1 hour, a large proportion of CXCR2 was re-expressed on the cell
surface in the cells overexpressing EGFP-Rab11a-S20V (Figure 9C-D), but
not in the cells overexpressing EGFP-Rab11a-S25N (Figure 9G-H). In
Figure 9C, the small arrows indicate that CXCR2 was recycled back to
the cell surface in cells overexpressing EGFP-Rab11a-S20V. In Figure
9G, the large arrow indicates that CXCR2 was retained in the cytoplasm
in cells overexpressing EGFP-Rab11a-S25N. As an internal control in
Figure 9G, in the cell without overexpression of EGFP-Rab11a-S25N
(without arrow), the receptor was mostly recycled back to the cell
surface. These data strongly suggest regulation of CXCR2 recycling by
Rab11a.
Rab7 is required for the transport of CXCR2 to lysosomes We have demonstrated in Figure 5 that CXCR2 was colocalized with LDL in response to agonist treatment for 4 hours, suggesting that a proportion of CXCR2 was transported to lysosomes. To determine whether Rab7 is involved in lysosomal sorting of CXCR2, HEK293 cells stably coexpressing CXCR2 and the wild-type Rab7 or the dominant-negative mutant Rab7-T22N were treated with CXCL8 (200 nM) for 4 hours, and the colocalization of CXCR2 with Lamp-1 was determined by confocal microscopy. CXCR2 was colocalized with LAMP-1 in the control cells (Figure 10A), which is consistent with the colocalization of the receptor with LDL shown in Figure 5. Interestingly, the colocalization of CXCR2 with Lamp-1 appeared to be increased in the cells overexpressing wild-type Rab7 proteins (Figure 10B). In contrast to wild-type Rab7, which did not change the perinuclear aggregation of lysosomes (Figure 10B), overexpressing a dominant-negative mutant Rab7-T22N significantly changed the morphology of Lamp-1 staining. Lamp-1 staining became smaller and was dispersed throughout the cytoplasm in cells overexpressing Rab7-T22N, as shown in Figure 10C, and the colocalization of CXCR2 with Lamp-1 was significantly decreased (Figure 10C). Rab7-T22N has been shown to block the lysosomal sorting of LDL for degradation,41 so that the colocalization of LDL with LAMP-1 in the Rab7- or the Rab7-T22N-expressing cells could be considered as a positive control for the effect of Rab7 on the transport of CXCR2 to lysosomes. Cells were loaded with Dil-LDL for 4 hours, and the colocalization of LDL with LAMP-1 was examined by confocal microscopy. As expected, LDL strongly colocalized with LAMP-1 in the cells overexpressing Rab7 (Figure 10D), but the colocalization was remarkably reduced in the cells overexpressing Rab7-T22N (Figure 10E). These data suggest that CXCR2 is targeted to degradation in lysosomes after prolonged agonist treatment and that Rab7 plays a role in the transport of the receptor to lysosomes.
The above data suggest that CXCR2 is transported from early endosomes
to late endosomes/lysosomes. Thus, blocking lysosomal sorting by
overexpressing the dominant-negative mutant Rab7-T22N may result in
retention of the receptors in the early endosomes. In addition, because
CXCR2 may traffic from early to recycling endosomes, blocking the
lysosomal sorting may also result in the retention of some receptors in
the recycling endosomes. To test this hypothesis, HEK293 cells stably
coexpressing CXCR2 and Rab7 or Rab7-T22N were transiently transfected
with cDNA for EGFP-Rab5 or EGFP-Rab11a. Cells were treated with CXCL8
for 4 hours, when most of the internalized receptors have left these 2 Rab protein-positive endosomes for late endosomes/lysosomes (Figures
1D, 2D, 3D). As anticipated, CXCR2 exhibited little colocalization with
EGFP-Rab5 and EGFP-Rab11 in the cells overexpressing wild-type Rab7
(Figure 11A, C) following CXCL8
treatment for 4 hours. However, the colocalization of CXCR2 and
EGFP-Rab5 or EGFP-Rab11a was remarkably increased in the cells
overexpressing Rab7-T22N (Figure 11B,D). These data suggest that
blocking late endosomal/lysosomal sorting by overexpressing Rab7-T22N
results in the retention of the receptor in early and recycling
endosomes.
LDL is transported quickly from early endosomes to late endosomes/lysosomes after uptake.39 To examine whether the overexpression of Rab7-T22N results in the retention of LDL in early endosomes, HEK293 cells stably expressing Rab7 or Rab7-T22N were transfected with EGFP-Rab5. Cells were loaded with Dil-LDL (40 µg/mL) for 4 hours, and the colocalization of Dil-LDL with EGFP-Rab5 was determined by confocal microscopy. As anticipated, there was little colocalization between LDL and EGFP-Rab5 in the Rab7-expressing cells (Figure 11E). However, in the Rab7-T22N-expressing cells, LDL exhibited significantly increased colocalization with the EGFP-Rab5 (Figure 11F). These data, taken together with the effect of Rab7-T22N on the morphologic alteration of lysosomal compartments (Figure 10C,E), suggest that Rab7-T22N causes the disorganization of lysosomes, thus blocking protein transport from early endosomes to lysosomes.
To gain insight into the intracellular trafficking pathway of CXCR2 and to determine the fine-tuning of the receptor transport among endosomal compartments, we focused on the regulation of CXCR2 trafficking by various Rab GTPases, which have been implicated principally in the control of membrane budding, transport, vesicle docking, and membrane fusion.37 CXCR2 is expressed predominantly in neutrophils, but, because of the technical difficulties in transfection and confocal microscopy using the suspended neutrophils, we used a well-characterized model system (HEK293 cells) in which the receptor and the EGFP-tagged Rab GTPases are overexpressed. This system was shown to highly resemble neutrophils in ligand-binding affinity, receptor-G-protein coupling, homologous desensitization, internalization, and recycling of CXCR2.12,13,42-44 The present study demonstrated that multiple Rab GTPases are involved in the intracellular trafficking of CXCR2 in HEK293 cells. To provide evidence that these Rab proteins play the same role in the receptor trafficking in some other cell lines, we also determined the colocalization of CXCR2 with these Rab proteins in RBL-2H3 cells, which are widely used for the study of chemokine receptors. Endocytosis is characterized by vesicular transport along numerous pathways. Common steps in each pathway include membrane budding to form vesicles, transport to a particular destination, and ultimately docking and fusion with the target membrane. Agonist-bound CXCR2 appears to internalize into vesicles through a dynamin-mediated formation of clathrin-coated pits,13 after which the receptors are rapidly transported to early and recycling endosomal compartments. This is evident by the colocalization of the receptor with Rab5,29 Rab11a,23 and transferrin.38 However, prolonged agonist exposure results in transport of the receptor to late endosomes and lysosomes, as can be seen by the colocalization of the receptor with Rab7, LDL, and LAMP-1. CXCR2 internalization appears to be regulated by Rab5, based on the
data that overexpression of a dominant-negative mutant Rab5-S34N
attenuated the internalization of the ligand-bound CXCR2. Rab5 is
important for sequestering receptors into clathrin-coated pits,
subsequent fusion of vesicles with early endosomes, and homotypic
fusion between early endosomes.45,46 Rab5-S34N likely prevents CXCR2 endocytosis before pinching off of the clathrin-coated vesicles from the plasma membrane. We also observed that internalized CXCR2 was colocalized with the constitutively active mutant Rab5-Q79L. In contrast to the D2 dopamine receptor,33 the
overexpression of Rab5-Q79L did not significantly facilitate the
internalization of CXCR2 as measured by radioligand-binding assay.
These data are consistent with the report demonstrating that the
overexpression of Rab5-Q79L did not enhance CXCR2 has been demonstrated to colocalize with Rab11a on agonist
exposure.47 An interesting observation in this study is that CXCR2 was colocalized with Rab11a in the same time-frame as the
colocalization of the receptor with Rab5. Rab11a, which is concentrated
in recycling endosomes, was initially demonstrated to be important for
transferrin transport through recycling endosomes in nonpolarized
cells.48,49 The small effect of the dominant-negative mutant of Rab11a on CXCR2 internalization excludes the possibility of a
role for Rab11a in the early endosomal trafficking of CXCR2. Even
though CXCR2 exhibited little recycling in the continued presence of
agonist, it is recycled back to the cell membrane after removal of the
agonist and recovery of the cells at 37°C. Receptor recycling was
inhibited by overexpression of a dominant-negative mutant, Rab11a-S25N,
suggesting the regulatory role of Rab11a in CXCR2 recycling. Another
Rab protein that is localized in recycling endosomes is
Rab4.23 It has been postulated that Rab4 plays a role in
the recycling of Previous studies have demonstrated that prolonged agonist stimulation induces degradation of CXCR2.9,13,24 The inhibition of CXCR2 degradation by the lysosomal inhibitor chloroquine (data not shown) suggests that CXCR2 is degraded in lysosomes. We observed a dramatic increase in the colocalization of CXCR2 with Rab7 in response to prolonged agonist stimulation (4 hours). In the meantime, CXCR2 was found to be colocalized with Lamp-1 and LDL. These data suggest that most of the internalized CXCR2 is transported from early endosomes to late endosomes/lysosomes on prolonged ligand stimulation. In addition, studies on CXCR4 trafficking also demonstrated that CXCR4 colocalized with Lamp-1 after 3 hours of CXCL12 treatment.50 It is unclear why it takes longer for lysosomal sorting of chemokine receptors than of LDL. Rab7 likely plays an important role in the transport of CXCR2 to lysosomes. The inhibition of lysosomal sorting of CXCR2 by the overexpression of the dominant-negative mutant Rab7-T22N could be explained in 2 different ways. One interpretation is that Rab7 plays a role in the transport of the internalized receptor from late endosomes to lysosomes, which is hypothesized by Meresse et al51 based on their observation that a GTPase-defective Rab7 mutant (constitutively active) exhibits enhanced association with lysosomes. In contrast to the cells expressing the wild-type Rab7, which showed an aggregation of lysosomes in the perinuclear region, in the cells overexpressing the dominant-negative Rab7-T22N mutant, perinuclear lysosome aggregation disappeared and lysosomes identified by Lamp-1 became dispersed throughout the cytoplasm. Thus, Rab7 is considered to control the aggregation and fusion of late endocytic structures/lysosomes, which is essential for maintenance of the perinuclear lysosome compartment.52 The second interpretation is that Rab7 is essential for the transport from early to late endosomes.53 This hypothesis is supported by our observation that overexpression of the dominant-negative Rab7-T22N mutant, which blocks the transport of CXCR2 to lysosomes, resulted in the retention of CXCR2 in the Rab5-positive early endosomes. In addition, because there is a pathway from early to recycling endosomes, it is anticipated that overexpression of the dominant-negative Rab7-T22N mutant resulted in the localization of CXCR2 in recycling endosomes. Taken together, this study clearly demonstrated that the chemokine receptor CXCR2 undergoes a recycling pathway and lysosomal sorting pathways under different conditions. CXCR2 is sequestered into early endosomes on agonist exposure. The receptors are delivered to recycling endosomes and are partially recycled back to the cell membrane after removal of the agonist, though the recycling rate is very slow.54 In the prolonged presence of a saturated concentration of agonist, CXCR2 is transported predominantly to lysosomes for degradation, but this process is relatively slow compared with the quick lysosomal sorting of LDL.39 These data are important for understanding the regulation of the receptor functions under physiological and pathologic conditions. It is well known that one of the most important functions of chemokine receptors is to mediate chemotaxis of neutrophils and lymphocytes to the inflammatory sites, and it is postulated that receptor internalization and recycling play an important role in this process.12,13,55 Neutrophils and lymphocytes migrate to the inflammatory sites along the gradient concentrations of chemokines. When the cells are far away from the inflammatory sites where the chemokine concentrations are relatively low, some internalized chemokine receptors are able to undergo recycling and resensitization, so as to respond to the continued stimulation of the ligands for chemotaxis. When the cells reach the inflammatory sites, chemokine receptors are targeted for degradation in lysosomes without recycling because of the exposure of high concentrations of ligands. These result in termination of the chemotactic response and accumulation of neutrophils and lymphocytes in the inflammatory sites.
We thank Dr Angela Wandinger-Ness in the University of New Mexico Health Sciences Center for the generous gifts of the wild-type and mutant Rab7 plasmids, Dr Stephan S. G. Ferguson of the University of Western Ontario, London, ON, Canada, for the generous gifts of the wild-type and mutant Rab5 plasmids; Dr Richard Roberts of Vanderbilt University for the generous gifts of transferrin-Texas Red and Dil-LDL; and Dr Ricardo Richard of Meharry Medical College for offering us the RBL-2H3 cells. We also thank Dr Sam Wells of Vanderbilt University Ingram-Cancer Center for confocal analysis, and we thank Catherine Allen, Lynn Butler, and Melanie Smith of Nashville VA Medical Center for their assistance with FACS analysis. In addition, we thank Linda W. Horton and Yingchun Yu in our laboratory for technical help.
Submitted July 2, 2002; accepted October 25, 2002.
Prepublished online as Blood First Edition Paper, October 31, 2002; DOI 10.1182/blood-2002-07-1965.
Supported by a Department of Veterans Affairs career scientist grant (A.R.), National Cancer Institute grant CA 34590 (A.R.), Vanderbilt-Ingram Cancer Center support grant CA68485, and National Institutes of Health grants DK48370 (J.R.G.) and DK43405 (J.R.G.).
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: Ann Richmond, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; e-mail: ann.richmond{at}vanderbilt.edu.
1.
Luster AD.
Chemokines
2.
Proudfoot AE, Wells TN, Clapham PR.
Chemokine receptors 3. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635-638[CrossRef][Medline] [Order article via Infotrieve].
4.
Murphy PM, Baggiolini M, Charo IF, et al.
International union of pharmacology, XXII: nomenclature for chemokine receptors.
Pharmacol Rev.
2000;52:145-176 5. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity. 2000;12:121-127[CrossRef][Medline] [Order article via Infotrieve].
6.
Belperio JA, Keane MP, Arenberg DA, et al.
CXC chemokines in angiogenesis.
J Leukoc Biol.
2000;68:1-8 7. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol. 1999;17:657-700[CrossRef][Medline] [Order article via Infotrieve].
8.
Wu D, LaRosa GJ, Simon MI.
G-protein-coupled signal transduction pathways for interleukin-8.
Science.
1993;261:101-103
9.
Mueller SG, White JR, Schraw WP, Lam V, Richmond A.
Ligand-induced desensitization of the human CXC chemokine receptor-2 is modulated by multiple serine residues in the carboxyl-terminal domain of the receptor.
J Biol Chem.
1997;272:8207-8214
10.
Haribabu B, Richardson RM, Fisher I, et al.
Regulation of human chemokine receptors CXCR4: role of phosphorylation in desensitization and internalization.
J Biol Chem.
1997;272:28726-28731
11.
Orsini MJ, Parent JL, Mundell SJ, Benovic JL, Marchese A.
Trafficking of the HIV coreceptor CXCR4: role of arrestins and identification of residues in the C-terminal tail that mediate receptor internalization.
J Biol Chem.
1999;274:31076-31086 12. Fan GH, Yang W, Wang XJ, Qian Q, Richmond A. Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization. Biochemistry. 2001;40:791-800[CrossRef][Medline] [Order article via Infotrieve].
13.
Yang W, Wang D, Richmond A.
Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction.
J Biol Chem.
1999;274:11328-11333
14.
Aragay AM, Mellado M, Frade JM, et al.
Oncocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G-protein-coupled receptor kinase 2.
Proc Natl Acad Sci U S A.
1998;95:2985-2998 15. Aramori I, Ferguson SS, Bieniasz PD, Zhang J, Cullen B, Cullen MG. Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J. 1997;16:4606-4616[CrossRef][Medline] [Order article via Infotrieve].
16.
Barlic J, Khandaker MH, Mahon E, et al.
Beta-arrestins regulate interleukin-8-induced CXCR1 internalization.
J Biol Chem.
1999;274:16287-16294
17.
Cheng ZJ, Zhao J, Sun Y, et al.
Beta-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between beta-arrestin and CXCR4.
J Biol Chem.
2000;275:2479-2485
18.
Fan GH, Yang W, Sai J, Richmond A.
Phosphorylation-independent association of CXCR2 with the protein phosphatase 2A core enzyme.
J Biol Chem.
2001;276:16960-16968
19.
Asagoe K, Yamamoto K, Takahashi A, et al.
Down-regulation of CXCR2 expression on human polymorphonuclear leukocytes by TNF-
20.
Khandaker MH, Xu L, Rahimpour R, et al.
CXCR1 and CXCR2 are rapidly down-modulated by bacterial endotoxin through a unique agonist-independent, tyrosine kinase-dependent mechanism.
J Immunol.
1998;161:1930-1938
21.
Brandt SM, Mariani R, Holland AU, Hope TJ, Landau NR.
Chemokine-mediated block to HIV entry is associated with CCR5 internalization efficiency.
J Biol Chem.
2002;277:17291-17299
22.
Daro E, van der Sluijs P, Galli T, Mellman I.
Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.
Proc Natl Acad Sci U S A.
1996;93:9559-9564 23. Mohrmann K, van der Sluijs P. Regulation of membrane transport through the endocytic pathway by rab GTPases. Mol Membr Biol. 1999;16:81-87[CrossRef][Medline] [Order article via Infotrieve]. 24. Bucci C, Parton RG, Mather IH, et al. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992;70:715-728[CrossRef][Medline] [Order article via Infotrieve].
25.
Zuk PA, Elferink LA.
Rab15 mediates an early endocytic event in Chinese hamster ovary cells.
J Biol Chem.
1999;274:22303-22312
26.
Chen W, Feng Y, Chen D, Wandinger-Ness A.
Rab11 is required for trans-Golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor.
Mol Biol Cell.
1998;9:3241-3257 27. Duman JG, Tyagarajan K, Kolsi MS, Moore HP, Forte JG. Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase. Am J Physiol. 1999;277:C361-C372[Medline] [Order article via Infotrieve].
28.
Casanova JE, Wang X, Kumar R, et al.
Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells.
Mol Biol Cell.
1999;10:47-61 29. Chavrier P, Parton RG, Hauri HP, Simons K, Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990;62:317-329[CrossRef][Medline] [Order article via Infotrieve]. 30. Lombardi D, Soldati T, Riederer MA, Goda Y, Zerial M, Pfeffer SR. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J. 1993;12:677-682[Medline] [Order article via Infotrieve].
31.
Seachrist JL, Anborgh PH, Ferguson SS.
Beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases.
J Biol Chem.
2000;275:27221-27228 32. Moore RH, Sadovnikoff N, Hoffenberg S, et al. Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes. J Cell Sci. 1995;108:2983-2991[Abstract]. 33. Iwata K, Ito K, Fukuzaki A, Inaki K, Haga T. Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors. Eur J Biochem. 1999;263:596-602[Medline] [Order article via Infotrieve].
34.
Mueller SG, Schraw WP, Richmond A.
Activation of protein kinase C enhances the phosphorylation of the type B interleukin-8 receptor and stimulates its degradation in non-hematopoietic cells.
J Biol Chem.
1995;270:10439-10448
35.
Lapierre LA, Kumar R, Hales CM, et al.
Myosin vb is associated with plasma membrane recycling systems.
Mol Biol Cell.
2001;12:1843-1857
36.
Feng Y, Press B, Wandinger-Ness A.
Rab 7: an important regulator of late endocytic membrane traffic.
J Cell Biol.
1995;131:1435-1452 37. Somsel Rodman J, Wandinger-Ness A. Rab GTPases coordinate endocytosis. J Cell Sci. 2000;113:183-192[Abstract]. 38. Stenmark H, Paarton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 1994;13:1287-1296[Medline] [Order article via Infotrieve]. 39. Li Y, Cam J, Bu G. Low-density lipoprotein receptor family: endocytosis and signal transduction. Mol Neurobiol. 2001;23:53-67[CrossRef][Medline] [Order article via Infotrieve]. 40. Ray E, Samanta AK. Receptor-mediated endocytosis of IL-8: a fluorescent microscopic evidence and implication of the process in ligand-induced biological response in human neutrophils. Cytokine. 1997;9:587-596[CrossRef][Medline] [Order article via Infotrieve].
41.
Vitelli R, Santillo M, Daniela L, et al.
Role of the small GTPase RAB7 in the late endocytic pathway.
J Biol Chem.
1997;272:4391-4397 42. Ben-Baruch A, Grimm M, Bengali K, et al. The differential ability of IL-8 and neutrophil-activating peptide-2 to induce attenuation of chemotaxis is mediated by their divergent capabilities to phosphorylate CXCR2 (IL-8 receptor B). J Immunol. 1997;158:5927-5933[Abstract]. 43. Feniger-Barish R, Ran M, Zaslaver A, BenBaruch A. Differential modes of regulation of CXC chemokine-induced internalization and recycling of human CXCR1 and CXCR2. Cytokine. 1999;11:996-1009[CrossRef][Medline] [Order article via Infotrieve].
44.
Ben-Baruch A, Bengali KM, Biragyn A, et al.
Interleukin-8 receptor beta: the role of the carboxyl terminus in signal transduction.
J Biol Chem.
1995;270:9121-9128 45. Christoforidis S, Miaczynska M, Ashman K, et al. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat Cell Biol. 1999;1:249-252[CrossRef][Medline] [Order article via Infotrieve]. 46. McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe EA. Novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr Biol. 1998;8:34-45[CrossRef][Medline] [Order article via Infotrieve].
47.
Feniger-Barish R, Belkin D, Zaslaver A, et al.
GCP-2-induced internalization of IL-8 receptors: hierarchical relationships between GCP-2 and other ELR(+)-CXC chemokines and mechanisms regulating CXCR2 internalization and recycling.
Blood.
2000;95:1551-1559
48.
Ren M, Xu G, Zeng J, De Lemos-Chiarandini C, Adesnik M, Sabatini DD.
Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes.
Proc Natl Acad Sci U S A.
1998;95:6187-6192
49.
Ullrich O, Reinsch S, Urbe S, Zerial M, Parton RG.
Rab11 regulates recycling through the pericentriolar recycling endosome.
J Cell Biol.
1996;135:913-924
50.
Marchese A, Benovic JL.
Agonist-promoted ubiquitination of the G-protein-coupled receptor CXCR4 mediates lysosomal sorting.
J Biol Chem.
2001;276:45509-45512 51. Meresse S, Gorvel JP, Chavrier P. The rab7 GTPase resides on a vesicular compartment connected to lysosomes. J Cell Sci. 1995;108:3349-3358[Abstract].
52.
Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B.
Rab7: a key to lysosome biogenesis.
Mol Biol Cell.
2000;11:467-480
53.
Mukhopadhyay A, Funato K, Stahl PD.
Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes.
J Biol Chem.
1997;272:13055-13059 54. Chuntharapai A, Kim KJ. Regulation of the expression of IL-8 receptor A/B by IL-8: possible functions of each receptor. J Immunol. 1995;155:2587-2594[Abstract].
55.
Fan G-H, Yang W, Sai J-Q, Richmond A.
Hsc/Hsp interacting protein (Hip) associates with CXCR2 and regulates the receptor signaling and trafficking.
J Biol Chem.
2002;277:6590-6597
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  Y.-W. Huang, P. Su, G. Y. Liu, M. R. Crow, D. Chaukos, H. Yan, and L. A. Robinson Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases J. Biol. Chem., October 23, 2009; 284(43): 29644 - 29653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Jones and P. M. Hinkle Subcellular Trafficking of the TRH Receptor: Effect of Phosphorylation Mol. Endocrinol., September 1, 2009; 23(9): 1466 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Stillie, S. M. Farooq, J. R. Gordon, and A. W. Stadnyk The functional significance behind expressing two IL-8 receptor types on PMN J. Leukoc. Biol., September 1, 2009; 86(3): 529 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Baugher and A. Richmond The Carboxyl-terminal PDZ Ligand Motif of Chemokine Receptor CXCR2 Modulates Post-endocytic Sorting and Cellular Chemotaxis J. Biol. Chem., November 7, 2008; 283(45): 30868 - 30878. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Garcia-Regalado, M. L. Guzman-Hernandez, I. Ramirez-Rangel, E. Robles-Molina, T. Balla, J. Vazquez-Prado, and G. Reyes-Cruz G Protein-coupled Receptor-promoted Trafficking of G{beta}1{gamma}2 Leads to AKT Activation at Endosomes via a Mechanism Mediated by G{beta}1{gamma}2-Rab11a Interaction Mol. Biol. Cell, October 1, 2008; 19(10): 4188 - 4200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yu, Y. Su, S. R. Opalenik, T. Sobolik-Delmaire, N. F. Neel, S. Zaja-Milatovic, S. T. Short, J. Sai, and A. Richmond Short tail with skin lesion phenotype occurs in transgenic mice with keratin-14 promoter-directed expression of mutant CXCR2 J. Leukoc. Biol., August 1, 2008; 84(2): 406 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Wang, X. Chen, X. Zhang, and L. Ma Phosphorylation State of {micro}-Opioid Receptor Determines the Alternative Recycling of Receptor via Rab4 or Rab11 Pathway Mol. Endocrinol., August 1, 2008; 22(8): 1881 - 1892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 B. X. Li, A. K. Satoh, and D. F. Ready Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors J. Cell Biol., May 21, 2007; 177(4): 659 - 669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. F. Neel, L. A. Lapierre, J. R. Goldenring, and A. Richmond RhoB plays an essential role in CXCR2 sorting decisions J. Cell Sci., May 1, 2007; 120(9): 1559 - 1571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Lapierre, K. M. Avant, C. M. Caldwell, A.-J. L. Ham, S. Hill, J. A. Williams, A. J. Smolka, and J. R. Goldenring Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1249 - G1262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. L. Rittner, D. Labuz, M. Schaefer, S. A. Mousa, S. Schulz, M. Schafer, C. Stein, and A. Brack Pain control by CXCR2 ligands through Ca2+-regulated release of opioid peptides from polymorphonuclear cells FASEB J, December 1, 2006; 20(14): 2627 - 2629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Luo, H. Pan, M. Mines, K. Watson, J. Zhang, and G.-H. Fan CXCL12 Induces Tyrosine Phosphorylation of Cortactin, Which Plays a Role in CXC Chemokine Receptor 4-mediated Extracellular Signal-regulated Kinase Activation and Chemotaxis J. Biol. Chem., October 6, 2006; 281(40): 30081 - 30093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Ducharme, C. M. Hales, L. A. Lapierre, A.-J. L. Ham, A. Oztan, G. Apodaca, and J. R. Goldenring MARK2/EMK1/Par-1B{alpha} Phosphorylation of Rab11-Family Interacting Protein 2 Is Necessary for the Timely Establishment of Polarity in Madin-Darby Canine Kidney Cells Mol. Biol. Cell, August 1, 2006; 17(8): 3625 - 3637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Rittner, S. A. Mousa, D. Labuz, K. Beschmann, M. Schafer, C. Stein, and A. Brack Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain J. Leukoc. Biol., May 1, 2006; 79(5): 1022 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Ding, M. Li, J. Zhang, N. Li, Z. Xia, Y. Hu, S. Wang, and G.-H. Fan The 73-kDa Heat Shock Cognate Protein Is a CXCR4 Binding Protein that Regulates the Receptor Endocytosis and the Receptor-Mediated Chemotaxis Mol. Pharmacol., April 1, 2006; 69(4): 1269 - 1279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vidricaire and M. J. Tremblay Rab5 and Rab7, but Not ARF6, Govern the Early Events of HIV-1 Infection in Polarized Human Placental Cells J. Immunol., November 15, 2005; 175(10): 6517 - 6530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Hamelin, C. Theriault, G. Laroche, and J.-L. Parent The Intracellular Trafficking of the G Protein-coupled Receptor TP{beta} Depends on a Direct Interaction with Rab11 J. Biol. Chem., October 28, 2005; 280(43): 36195 - 36205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Luo, Y. Ding, K. Watson, J. Zhang, and G.-H. Fan N-Methyl-D-aspartate Attenuates CXCR2-Mediated Neuroprotection through Enhancing the Receptor Phosphorylation and Blocking the Receptor Recycling Mol. Pharmacol., August 1, 2005; 68(2): 528 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Gebhardt, U. Breitenbach, K. H. Richter, G. Furstenberger, C. Mauch, P. Angel, and J. Hess c-Fos-Dependent Induction of the Small Ras-Related GTPase Rab11a in Skin Carcinogenesis Am. J. Pathol., July 1, 2005; 167(1): 243 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. S. Hall, E. Smith, W. Langer, L. A. Jacobs, D. Goulding, and M. C. Field Developmental Variation in Rab11-Dependent Trafficking in Trypanosoma brucei Eukaryot. Cell, May 1, 2005; 4(5): 971 - 980. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-O. Yoon, S. Shin, and A. M. Mercurio Hypoxia Stimulates Carcinoma Invasion by Stabilizing Microtubules and Promoting the Rab11 Trafficking of the {alpha}6{beta}4 Integrin Cancer Res., April 1, 2005; 65(7): 2761 - 2769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Hynes, S. M. Mervine, E. A. Yost, J. L. Sabo, and C. H. Berlot Live Cell Imaging of Gs and the {beta}2-Adrenergic Receptor Demonstrates That Both {alpha}s and {beta}1{gamma}7 Internalize upon Stimulation and Exhibit Similar Trafficking Patterns That Differ from That of the {beta}2-Adrenergic Receptor J. Biol. Chem., October 15, 2004; 279(42): 44101 - 44112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. Moore, E. E. Millman, E. Alpizar-Foster, W. Dai, and B. J. Knoll Rab11 regulates the recycling and lysosome targeting of {beta}2-adrenergic receptors J. Cell Sci., July 1, 2004; 117(15): 3107 - 3117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Rose, J. F. Foley, P. M. Murphy, and S. Venkatesan On the Mechanism and Significance of Ligand-induced Internalization of Human Neutrophil Chemokine Receptors CXCR1 and CXCR2 J. Biol. Chem., June 4, 2004; 279(23): 24372 - 24386. [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]
|
|
|
|
|
|
M. Weber, E. Blair, C. V. Simpson, M. O'Hara, P. E. Blackburn, A. Rot, G. J. Graham, and R. J.B. Nibbs The Chemokine Receptor D6 Constitutively Traffics to and from the Cell Surface to Internalize and Degrade Chemokines Mol. Biol. Cell, May 1, 2004; 15(5): 2492 - 2508. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-arrestin and adaptin 2.
) or LDL (
) was determined by counting the colocalized receptor
fluorescence (yellow) from the total internalized receptor fluorescence
(green and yellow). Data are means ± SEMs of 3 independent
experiments (**P < .01). Tf indicates
transferrin.
chemotactic cytokines that mediate inflammation.
N Engl J Med.
1998;12:436.
.
J Immunol.
1998;160:4518-4525