Western Blotting

Cells were lysed with 1% Triton X-100 in 150 mM NaCl, 50 mM HEPES, 0.1 M EGTA, 2 mM MgCl2, 10% glycerol containing leupeptin, aprotinin and pepstatin (1 µg/ml each, Roche, Basel, Switzerland). Proteins from total cell lysates were resolved by SDS-PAGE and transferred to Protran nitrocellulose membrane (Schleicher &

Schuell, Dassel, Germany). Membranes were blocked with PBS containing 5% of low-fat dry milk and incubated with the respective antibodies overnight at 4°C or for one h at room temperature on a rocking plate. After washing, HRP-conjugated secondary antibodies were bound and detected using enhanced chemiluminescence (Pierce/Socochim, Lausanne, Switzerland).

Chapter 3

CCR7 signalling and the possible mechanism of endocytosis

Carolina Otero, Petra Eisele, Marcus Groettrup and Daniel F. Legler

3.1. Abstract

CCR7 is a seven transmembrane domain G protein coupled receptor, which is activated by the binding of its ligands, the CC chemokines SLC (Secondary lymphoid-tissue chemokine, CCL21) and ELC (EBI1 Ligand Chemokine, CCL19).

CCR7 has been very well characterized in its central role in homing and trafficking of lymphocytes and dendritic cells (DCs) to secondary lymphoid tissues. However, there is only little information on how CCR7 is internalized after ligand binding and which signals and proteins are involved in the migration process. To overcome this lack of information, we generated several mutants to investigate a possible role of ubiquitin on endocytosis and migration. We also created mutants where part or the entire C-terminal tail of CCR7 was removed. In this work we demonstrate that CCR7 is not ubiquitylated and that CCR7 mutants lacking putative ubiquitylation sites at its C terminus behave normal in term of receptor endocytosis, recycling and in mediating cell migration. Interestingly, CCR7 lacking its whole C-terminal tail is expressed normally at the plasma membrane, and endocytozed similar to CCR7wt upon ligand binding. However, cells transfected with CCR7 mutant lacking its C-terminal tail were unable to migrate, clearly suggesting that the CCR7 C-terminus contains essential amino acids for signal transduction being independent of CCR7 endocytosis.

3.2. Introduction

Receptor internalization is a very well known process used by membrane receptors in order to regulate the expression on the cell surface and its activation.

Through this mechanism, cells are able to regulate receptor density at the cell membrane and, therefore alter the magnitude of response to ligand binding. Receptors can be internalized via different pathways. The best studied pathway relies on clathrin-coated pits where a complex machinery of different adaptor proteins is involved, and where two main consensus signals at the C-terminus of the receptor have also been described (Bonifacino and Traub, 2003; Traub, 2003; Kirchhausen, 2002). Most receptors are internalized via this pathway. Another mechanism of internalization is via caveolae-mediated endocytosis, but only few receptors are known to use this pathway (Rajendran and Simons, 2005; Parton and Richards, 2003;

Le Roy and Wrana, 2005). Alternatively, some other receptors are internalized independently of clathrin-coated pits or caveolae-mediated endocytosis (Damm et al., 2005). Some receptors can be internalized by all these different mechanisms, depending normally on the concentration of the ligand, like in the case of EGFR (Aguilar and Wendland, 2005; Stang et al., 2004; Sigismund et al., 2005).

One signal for endocytosis, leading to both clathrin dependent or independent endocytosis is through post-transcriptional modifications by ubiquitin (Raiborg et al., 2002) (Madshus, 2006; Marchese and Benovic, 2004). The ubiquitylation process normally takes place at the intracellular part of the receptor. Ubiquitin may be added only in a single copy, resulting in monoubiquitylation, or many times, where ubiquitin will form a lysine 63 linked protein chain, resulting in polyubiquitylation (Hoeller et al., 2006; Huang et al., 2006). A well characterized example for monoubiquitylation on chemokine receptor is CXCR4, which undergoes rapid agonist-promoted degradation in the lysosomes (Marchese and Benovic, 2001; Marchese et al., 2003).

The chemokine receptor CCR7 is a seven transmembrane domain, G protein coupled receptor expressed on mature dendritic cells (mDC) as well as on T, B lymphocytes and NK cells. This chemokine receptor is an important organizer of the primary immune response due to its central role in the correct homing of lymphocytes and mDC to secondary lymphoid organs (Sallusto et al., 1998; Stein et al., 2003).

Mice lacking CCR7 present delayed kinetics concerning antibody response and delayed type I hypersensitivity reactions and morphological abnormalities in all secondary lymphoid organs. This is due to an impaired migration of mDCs and lymphocytes, which fail to migrate into the draining lymph nodes (Forster et al., 1999). This process is guided by the two ligands for CCR7, both of the CC-chemokine family, ELC (CCL19) and SLC (CCL 21), which are selective agonists with similar affinities for CCR7 (Sullivan et al., 1999). The activation of this receptor mediated by these two chemokines triggers the re-organization of actin filaments, calcium influx, MAPK and Erk1/2 activation, resulting in cell migration towards the chemokine (Scandella et al., 2004; Sanchez-Sanchez, 2006).

In order to investigate which signals are involved in CCR7 endocytosis and migration, in order to have more information on the signalling pathway, we generated a CCR7 tagged with vsv at the N-terminus. Additionally, we generated several mutants, where possible ubiquitylation sites were mutated. In addition, we generated mutants where the C-terminal tail was entirely or only partially removed. Through the characterization of these mutants together with additional biochemical data we could show that CCR7 is not ubiquitylated even upon ligand binding. We could also observe that the endocytosis signal is not present within the C-terminus, however some amino acids in the C-terminal region were critical for signal transduction and migration, but not for endocytosis or recycling.

3.3. Results

3.3.1. Generation of functional vsvCCR7

In order to study the cell signalling complex and the process of endocytosis we generated a recombinant CCR7 with a N-terminal vsv tag (figure 3.1 A). Despite the correct sequence and surface staining with anti-CCR7 antibody (figure 3.1 B), we were not able to detect CCR7 via its vsv with antibodies against this tag. Probably this is due to a cleavage or a modification of this tag at the N-terminus. We generated this construct with the N-terminal tag with the objective to elucidate the cell signalling complex of the receptor. A C-terminal tag like in our other constructs (CCR7-GFP or CCR7-HA) may interfere with the coupling to other proteins.

To exclude the possibility that this could be a problem of the anti-vsv antibody, we made a western blot using a protein tagged with vsv (vsvTrail-R3), as a positive control. As shown in figure 3.1 C vsvTrail-R3 could be easily detected by an anti-vsv antibody, in contrast to vsvCCR7.

Figure 3.1. Characterization of vsvCCR7. (A) Scheme of vsvCCR7. (B) Cell surface expression of CCR7 was determined by flow cytometry using the monoclonal antibody 3D12 against CCR7 on untransfected HEK293 (blue line) or stably expressing vsvCCR7 (red line). (C) HEK293 cells transiently transfected with vsvTrail-R3 and stably transfected with vsvCCR7 were lysed with 1%

Triton X-100 lysis buffer. The lysate was loaded on an SDS-PAGE after centrifugation. A western blot with anti-vsv antibody was later performed.

This finding is unexpected as other chemokine receptors, such as CXCR4 can be tagged at the N-terminus (Marchese and Benovic, 2001). Further studies will be required to establish whether this is a problem concerning tag processing. Another possibility is that the signal peptide, which leads the protein to the plasma membrane, is situated at the N-terminus of CCR7, leaving no possibility to insert a tag at the very N-terminus.

3.3.2 Possible role of ubiquitylation for chemotaxis and CCR7 trafficking

Since no classical motifs for internalization have been found (Di-Leucine and NPXY motif) (Bonifacino and Traub, 2003), several mutants have been constructed in order to elucidate which amino acids might be important for CCR7 endocytosis.

CXCR4 was taken as a model receptor, whose trafficking relayed on mono-ubiquitylation. We analyzed the amino acid sequence of CCR7 in order to find C-terminal lysines where ubiquitin binding might occur, playing a role in the trafficking of CCR7. Four lysines in different intracellular loops have been found and three at the C-terminal tail (figure 3.2). Since ubiquitylation normally occurs at the C-terminus, we selected only the three lysines situated at the C-terminus for site-directed mutagenesis. To maintain the protein conformation, lysines were replaced by arginines keeping the positive charge, but do not allow ubiquitin conjugation.

Figure 3.2. Hypothetical scheme of CCR7. Mutated lysines are indicated in red. Other possible lysines that could be ubiquitylated are indicated in dashed red. At the terminus a putative site of N-glycosilation is shown.

Oligonucleotides containing the three mutations were synthesized and a PCR using CCR7-GFP as template together with the original CCR7 primers (CCR7se2 and CCR7as) was performed (figure 3.3). After this reaction two different PCR products were obtained. Both were denaturated and immediately a PCR was performed with the CCR7 primers, including restriction sites for cloning. This product was used to replace the insert of pcDNA3-CCR7wt by CCR7-3K3R.

Figure 3.3. Scheme of the site-directed mutagenesis. Lysines that were mutated are indicated with yellow thunders.

To characterize the possible role of ubiquitylation on chemotaxis or endocytosis, HEK293 and 300-19 cells were stably transfected with the mutant CCR7-3K3R-GFP. A chemotaxis assay was performed with 300-19 cells stably expressing CCR7wt, CCR7wt-GFP or CCR7-3K3R-GFP. Stably transfected cells with the mutated receptor normally migrated towards SLC and ELC, comparable to CCR7wt and CCR7wt-GFP (figure 3.4 A). This was also the case for receptor endocytosis where the receptor containing the mutation was internalized normally, even at a higher extent than CCR7wt-GFP (figure 3.4 B). Taken together, this data indicates that probably ubiquitylation at the C-terminus is neither required for cell migration nor for receptor internalization.

Figure 3.4. CCR7-3K3R-GFP is endocytozed and cells migrated normally towards both chemokines.

(A) Chemotaxis assay of 300-19 cells stably transfected with CCR7 wt, CCR7-GFP and CCR7-3K3R-GFP towards ELC and SLC (0,25µg/ml). As negative control a chemotaxis assay without chemokine was performed. (B) Endocytosis assay of HEK293 cells stably transfected with GFP and CCR7-3K3R-GFP. 100% surface expression corresponds to the amount of CCR7 at the surface under no chemokine conditions. Cells were incubated for 30 min at 37ºC and the total surface expression was measured with an anti-CCR7 antibody. Total fluorescence was analyzed by FACS.

To confirm the data and to exclude the possibility that this result is caused by the GFP-tag at the C-terminus of CCR7, GFP was replaced by an HA tag. Figure 3.5 shows a western blot, of HEK293T cells were transiently transfected with three different CCR7-3K3R-HA clones. 300-19 cells were transiently transfected with clone 1. A chemotaxis assay was performed, showing that cells expressing CCR7-3K3R-HA can migrate normally towards ELC. This confirms that the mutation itself does not affect the function of the receptor (data not shown).

Figure 3.5. Expression of CCR7-3K3R-HA. Western blot of HEK293 transiently transfected with different clones of CCR7-3K3R-HA or not transfected (WT). CCR7-HA was detected with an anti-HA antibody.

Studies performed by western blot with CCR7-HA or CCR7-3K3R-HA (data not shown and figure 3.5) have always shown CCR7 as a double band, in whole cell lysate as well as under immunoprecipitation conditions, leading us to assume that probably this is due to a possible ubiquitylation (in this mutant there are still four more lysines that remain unmutated) or to receptor glycosylation. To investigate this possibility, we analyzed the amino acid sequence of CCR7, where one possible site of glycosylation was found (figure 3.2), cells stably expressing CCR7-HA were treated (and left untreated) with tunicamycin, in order to block the glycosylation process.

Figure 3.6 shows a western blot of a CCR7-HA immunoprecipitation under both conditions, where we observed that the upper band of CCR7-HA disappears with tunicamycin treatment, confirming that this double band observed was due to receptor glycosylation and not to ubiquitylation.

Figure 3.6. CCR7 is glycosilated. 300-19 cells stably transfected with CCR7-HA were treated (and left untreated) for 4 hrs with Tunicamycin (50µg/ml). Cells were washed, lysed and from the lysate an immunoprecipitation was performed with anti-HA beads. Later proteins were resolved by SDS-PAGE and CCR7 was detected using an anti-HA antibody by western blot. Blue arrow indicates CCR7-HA.

Finally, we investigated in a direct approach the ubiquitylation of CCR7. To this end, we co-expressed CCR7-HA together with 3xFlag-ubiquitin following the protocol described by Marchese et al. (Marchese and Benovic, 2001). In brief, CCR7-HA was immunoprecipitated, followed by a western blot with anti-Flag antibody in

order to detect ubiquitin. As observed in figure 3.7 A, the first five lanes correspond to the total membrane lysate of the different transfection conditions. Lanes 1, 3, 4 and 5 show the total membrane protein that is ubiquitylated. The last five lanes show the immunoprecipitation with anti-HA beads (except lane 6 where the immunoprecipitation was performed with an irrelevant antibody). The lysate loaded in lane 9 was transfected with both HA and Ub-Flag showing no specific CCR7-ubiquitylation band. Following the example reported for CXCR4, cells transfected with both proteins were incubated with ELC for 30 min, to check whether CCR7 is ubiquitylated under these conditions or maybe even more after ligand stimulation.

However as it is shown in the last lane this was not the case since no band appeared at all. To confirm that CCR7-HA was properly transfected and that the immunoprecipitation worked we performed the same procedure as before, but this time an anti-HA antibody was used for western blot (figure 3.7 B).

To verify our working conditions, we performed the same experiment with HA-CXCR4 and Flag-Ub. Proper controls of transfection and immunoprecipitaion were also introduced, however no specific band for CXCR4-Ub was found.

Nevertheless, the data of the mutant together with our coimmunoprecipitation experiments suggest that CCR7 is not ubiquitylated. Further experiments showing a proper positive control of a membrane protein that is ubiquitylated will be necessary to confirm this data.

Figure 3.7. CCR7 is not ubiquitylated. (A) HEK293 cells stably transfected with CCR7-HA were transiently transfected with 3xFlag-Ubiquitin and Dynamin K44A. After 48 hrs, cells were lysed and sonicated. To obtain cell membranes the lysates were centrifuged and the pellet was incubated with HA beads overnight. The day after, beads were washed three times with PBS and the proteins were resolved in an SDS-PAGE. Afterwards a western blot was performed using an anti-flag antibody.

Lanes 1 to 5 correspond to membranes before immunoprecipitation. Lanes 6 to 10 correspond to immunoprecipitation with anti-HA beads, except for lane 6 were an unrelated antibody was used. Lane 1 is HEK293 stably transfected with CCR7-HA and transiently transfected with 3xFlag-Ubiquitin and Dynamin K44A. Lane 2 is HEK293 only with CCR7-HA. Lane 3 is only 3xFlag-Ubiquitin. Lane 4 1 is HEK293 stably transfected with CCR7-HA and transiently transfected with 3xFlag-Ubiquitin and Dynamin K44A. Lane 5 is the same as in lane 4 but before lysis, cells were incubated with ELC (2µg/ml) for 30 min at 37°C. Lane 6 is HEK293 stably transfected with CCR7-HA and transiently transfected with 3xFlag-Ubiquitin and Dynamin K44A, but immunoprecipitated with an unrelated antibody (anti-Myc). Lane 7 is HEK293 transfected only with CCR7-HA. Lane 8 is only 3xFlag-Ubiquitin. Lane 9 is HEK293 stably transfected with CCR7-HA and transiently transfected with 3xFlag-Ubiquitin and Dynamin K44A. Lane 10 is the same as in lane 9 but before lysis, cells were incubated with ELC for 30 min at 37°C. (B) Same experiment as before, but here the western blot was performed with an anti-HA antibody.

3.3.3 The CCR7 C-terminus is important for migration but not for endocytosis

Once the possibility that a putative signal for endocytosis is ubiquitylation was refuted, we generated three different deletions within the C-terminal region in order to detect, which amino acids play crucial roles in endocytosis and/or signalling. Figure 3.8 illustrates the three different mutants, all tagged with HA at the C-terminus. In MT-1 most of the C-terminal tail has been removed and the remaining lysine replaced by an arginine as shown in red. This has been made with the purpose of avoiding any possibility of ubiquitylation at the C-terminal part and to confirm our previous data.

The last 34 amino acids have been removed in the MT-2 mutant, whereas in MT-3 only the last 24 amino acids were deleted. Within these last 24 amino acids, the threonines and serines (shown in black) were reported to be phosphorylated after ligand binding (Kohout et al., 2004), indicating a possible role in receptor signalling and/or trafficking for these amino acids.

Figure 3.8. Scheme of the three CCR7 mutants. Arrows show the different sites of deletion within the C-terminus and where an HA tag has been inserted. In case of MT-1 a lysine has been replaced by an arginine, shown in red. Dark circles show possible sites of phosphorylation. Adapted from Kohout et al. (Kohout et al., 2004).

To characterize the properties of these mutants, 300-19 and HEK293 cells were stably transfected with each of the constructs. With 300-19 cells a chemotaxis assay towards both chemokines was performed (figure 3.9 A). MT-2 and MT-3 trasnfected cells migrated comparable to CCR7wt. Interestingly, MT-1 transfected cells did not migrate. After this finding, to investigate whether the receptor is still functional, calcium influx together with ERK activation after chemokine triggering was analyzed, showing that MT-1 was the only mutant where neither calcium influx nor ERK activation was found (data not shown). This indicates that amino acids between MT-1 and MT-2 are crucial for CCR7 signalling. Further site-directed mutations are required in order to localize precisely the amino acids responsible for this process.

Through microscopical and flow cytometric analysis, CCR7 expression at the plasma membrane was analyzed in the three mutants. Data showed no differences in the expression. The mutated receptors were able to reach the plasma membrane without being retained in the ER (data not shown). With this fact, trafficking of these mutants was analyzed through endocytosis/recycling assays. 300-19 cells stably expressing the three mutants were incubated with ELC or SLC for 30 min to internalize CCR7, and were then incubated with anti-CCR7 antibody or were left another hour at 37°C after washing in order to allow the receptor to recycle (figure 3.9 B). Upon ELC binding all CCR7 mutants were internalized showing the same pattern as CCR7wt. Similar result were obtained in recycling assays, where no difference between CCR7 mutants and CCR7wt was found (figure 3.9 B). To confirm this data the same experiment was performed in HEK293 cells stably transfected, which showed the same result as described before (data not shown).

To conclude these results, two important points can be made: firstly, endocytosis/recycling seems to be an independent process of chemotaxis. Secondly the signals for endocytosis must be situated in a intracellular loop, probably where the DRY motif is located, as has been reported for other chemokine receptors and other GPCRs (Arora et al., 1997; Hawtin, 2005).

Figure 3.9. Characterization of CCR7 mutants. (A) 300-19 cells stably expressing one of the mutated receptors or the wt respectively were subject to a Transwell^TM chemotaxis assay. Cells migrated towards ELC and SLC (0.25µg/ml) as well as cells in the input were counted by flow cytometry during an interval of 120s. As a control for spontaneous migration, 300-19 CCR7-HA cells were assayed without chemokine. Values represent the mean of three independent experiments ± SEM. (B) Receptor surface staining of 300-19 cells stably expressing CCR7-HA and the mutants respectively with an anti-CCR7 antibody were performed after endocytosis of either ligand (2µg/ml) or after allowing 1h recycling of the internalized receptor at 37ºC. Total fluorescence was detected by flow cytometry. Non-transfected cells were used as a control to show receptor independent effects. Values represent the mean of three independent experiments ± SEM. (nc: no chemokine).

Another way to answer the question whether endocytosis/recycling is required

Another way to answer the question whether endocytosis/recycling is required

Im Dokument Characterization of the chemokine receptor CCR7 and its role in cell migration (Seite 56-0)