Chapter 3 CCR7 signalling and the possible mechanism of endocytosis
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.