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First published online 29 July 2008
doi: 10.1242/jcs.029074

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Distinct motifs in the chemokine receptor CCR7 regulate signal transduction, receptor trafficking and chemotaxis

Carolina Otero^1,*, Petra S. Eisele^1,*, Karin Schaeuble^1,2,*, Marcus Groettrup^1,2 and Daniel F. Legler^2,3,

¹ Department of Biology, Division of Immunology, University of Konstanz, Konstanz, Germany
² Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Unterseestrasse 47, CH-8280 Kreuzlingen, Switzerland
³ Zukunftskolleg, University of Konstanz, Konstanz, Germany

View larger version (27K): [in this window] [in a new window]   Fig. 1. CCR7 C-terminal deletion mutants reach the plasma membrane. (A) Scheme of CCR7 and its C-terminal deletion mutants. The cytoplasmic C-terminus of CCR7 depicted is based on the prediction by Swiss-Prot (Swiss Institute of Bioinformatics, Basel, Switzerland). Amino acids according to accession number P32248 are indicated. MT1 to MT3 represent C-terminal truncation mutants of CCR7. In the case of MT1, a Lys was replaced by Arg. Potential phosphorylation sites (S, T) and potential ubiquitylation sites (K) are highlighted in bold. The conserved DRY motif in the second intracellular loop was mutated to DNY by site-directed mutagenesis. (B) CCR7 and its C-terminal deletion mutants are equally expressed at the plasma membrane. HEK293 cells stably expressing wild-type (wt) CCR7, MT1, MT2 or MT3 were stained with a FITC-conjugated CCR7-specific antibody and cell surface expression was monitored by flow cytometry. Dashed lines represent untransfected cells stained with the same antibody. FITC-labeled isotype control staining on transfected cells revealed the same results. The experiment has been repeated twice yielding similar results.

View larger version (32K): [in this window] [in a new window]   Fig. 2. CCL19 binds equally to all CCR7 variants. 300-19 pre-B cells stably transfected with wild-type CCR7 or the CCR7 mutants were incubated with monobiotinylated CCL19 (1 µg/ml) for 20 minutes at 4°C. CCR7-bound CCL19-biotin was then detected with streptavidin-FITC and monitored by flow cytometry (upper panel). CCR7 cell surface staining was performed with a FITC-labeled CCR7-specific antibody (lower panel). Dashed lines represent untransfected cells stained with the same antibody. FITC-labeled isotype control staining on transfected cells revealed the same results. Results are representative of two experiments.

View larger version (11K): [in this window] [in a new window]   Fig. 3. The membrane proximal part of the C-terminus and the DRY motif of CCR7 are crucial for cell migration. Chemotaxis of 300-19 cells stably transfected with wild-type CCR7, MT1-3 or DNY mutants was assessed in TranswellTM chemotaxis assays. After 3 hours of incubation at 37°C, cells migrated in response to graded concentrations of CCL19 (A) or CCL21 (B) to the lower chamber were collected and counted by flow cytometry. Mean values ± s.d. of three independent experiments are depicted as percentage of input cells.

View larger version (75K): [in this window] [in a new window]   Fig. 4. The CCR7 mutants MT1 and DNY are unable to mobilize intracellular free Ca2+ upon chemokine binding. The parental murine pre-B cell line 300-19 or cells stably expressing CCR7, or the CCR7 mutants MT1, MT2, MT3 or DNY were loaded with Fluo-3/AM and stimulated with 1 µg/ml CCL19 or CCL21, and chemokine-mediated changes in fluorescence due to mobilization of intracellular calcium were recorded over time by flow cytometry. As a control, cells were also stimulated with 20 ng/ml ionomycin. A representative experiment out of three is shown.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Erk1/2 phosphorylation is sensitive to B. pertussis toxin and is not achieved by triggering the CCR7 mutants MT1 and DNY. (A) MT1 triggering by CCL19 does not lead to Erk1/2 phosphorylation. HEK293 cells stably expressing CCR7 or the C-terminal truncations MT1-MT3 were incubated with 2 µg/ml CCL19 for 5 minutes. Western blots derived from total cell lysates were probed with a monoclonal antibody specific for phosphorylated Erk1/2 (pErk-1/2). The nitrocellulose membrane was stripped and reprobed with a polyclonal anti-total Erk2 (tErk-2) antibody as a loading control. (B) PTx treatment impairs CCR7 mediated Erk1/2 activation. HEK293 cells stably transfected with wild-type CCR7 were pretreated or not with 100 ng/ml PTx for 2 hours followed by stimulation with 2 µg/ml CCL19 for 5 minutes. Erk1/2 phosphorylation was analyzed by western blotting as in A. (C) DNY mutation abolishes chemokine-mediated Erk1/2 activation. 300-19 cells stably expressing wild-type CCR7 and the DNY mutant were left untreated or incubated with 2 µg/ml CCL19 for 5 minutes and Erk1/2 phosphorylation was analyzed as above. One out of at least three experiments with similar results is shown.

View larger version (6K): [in this window] [in a new window]   Fig. 6. The membrane proximal part of the C-terminus and the DRY motif of CCR7 are crucial for G-protein activation. Membranes of 300-19 cells stably transfected with expression constructs for CCR7 or one of the four receptor mutants were prepared and binding of [35S]GTPS was assessed in response to stimulation with CCL19 (5 µg/ml). Results are expressed as fold over basal [35S]GTPS binding in the absence of chemokine. Values represent the mean ± s.e.m. of three experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Endocytosis and recycling do not require motifs in the C-terminal tail of CCR7 and are independent of G-protein activation. (A) Removing the entire C-terminus of CCR7 does not abolish chemokine-mediated receptor trafficking. 300-19 cells stably expressing wild-type CCR7 or C-terminal truncation mutants were stimulated with 2 µg/ml CCL19 or medium for 30 minutes at 37°C, followed by detection of CCR7 surface expression by flow cytometry using a CCR7 antibody to determine endocytosis (black bars). To analyze receptor recycling, chemokine-triggered cells were washed extensively to remove unbound CCL19 and subsequently incubated for 1 hour in the absence of chemokines, permitting recycling of CCR7 back to the plasma membrane (white bars). Relative CCR7 surface expression as a percentage of untreated cells is plotted. (B) CCR7 trafficking is PTx insensitive. 300-19 CCR7 cells were pretreated or not with PTx for 2 hours, followed by stimulation with CCL19. CCR7 internalization and recycling were determined by flow cytometry as described above. (C) The DRY motif is dispensable for CCR7 trafficking. 300-19 cells stably expressing wild-type CCR7 or the DNY mutant were incubated with 2 µg/ml CCL19 for 30 minutes for internalization assays. CCR7 endocytosis and recycling were assessed as outlined in A. Values represent the mean ± s.e.m. of three experiments.

View larger version (7K): [in this window] [in a new window]   Fig. 8. PKC plays only a minor role in CCR7 endocytosis. 300-19 CCR7 cells were pretreated or not with 10 µM PKC inhibitor Bim for 30 minutes and stimulated with either 1 µM PMA or 2 µg/ml CCL19 for 30 minutes at 37°C as indicated. Surface expression of CCR7 was then determined by flow cytometry. Relative mean median fluorescence ± s.e.m. of four experiments is shown.

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