Figure 1.
Interaction of DRiP78 with chemokine receptor dimmers.
HEK293 cells were co-transfected for 48 hours with various contructs harboring non-functional parts of a YFP variant, venus. Upon interaction between two receptors, the venus parts can associate together and regenerate a functional fluorescent signal. Receptor pairs were expressed with DRiP78-Rluc and bioluminescence resonance energy transfer was measured. * = p<0.05 compared with controls.
Figure 2.
DRiP78 interaction with CCR5 homodimers.
a) CXCR4 and CCR5 sequences corresponding to the potential interaction site of DRiP78, starting at the beginning of the carboxy-terminal tail of the receptor. b) Co-immunoprecipitation of DRiP78 with CCR5 WT or with a mutation of the potential interaction site (phenylalanine residues mutated to alanine residues) and with CXCR4-v1/CCR5-v2. c) Interaction of CXCR4 with DRiP78. d) CCR5 c-tail interaction with DRiP78. The GST-CCR5 c-tails (wild type or F/A mutant) were incubated HEK293A cell lysates expressing DRiP78 to detect the interaction.
Figure 3.
Effect of DRiP78 on CCR5 localization.
Cells were plated on coverslips for 24 hours and then transfected with HA-CCR5 (WT (a) or F/A mutant (b)) and a cell surface marker, the CB1 receptor. c) expression of WT CCR5 with DRiP78 and d) Expression of mutated CCR5 with DRiP78. Green shows HA expression, red shows CB1 receptor or FLAG expression while the overlay column shows merged images of the previous two as acquired via fluorescence microscopy. DRiP78 is an ER chaperone, and therefore serves as an ER marker.
Figure 4.
Effect of DRiP78 on Chemokine receptor plasma membrane localization.
a) Knockdown levels of DRiP78 expression with the DRiP78-targetting shRNA. 48 hours post-transfection with cDNAs encoding DRiP78 (WT or shRNA) and receptor constructs forming CCR5 homodimers or CXCR4-CCR5 heterodimers, cells were labeled with biotin and precipitated using streptavidin. An immunoblot against GFP was then performed to show receptor levels at plasma membrane. c, and d) Quantification of at least 3 experiments, as performed in b). e) Plasma membrane expression of CCR5 WT or F/A in presence of DRiP78 WT or shRNA. f) Fluorescence measurements of cell surface expression of HA-CCR5 WT and F/A using an ALEXA-488 coupled secondary antibody.
Figure 5.
DRiP78 levels in cells affect function.
a) Increasing amounts of DRiP78 cDNA were transfected in HEK293 cells and expression levels of chemokine receptor dimers were measured. b) Cells expressing chemokine receptor dimers were transfected for 48 hours with various amounts of cDNA encoding DRiP78 and fluorescence levels were then measured. Fluorescence appears only when receptors dimerize therefore any variation in fluorescence level would be attributed to a change in the capacity of those receptors to associate. c) Quantification of Rluc levels in cells, when expressed along with constructs mentioned in b). Rluc levels do not change, so fluorescence levels are not attributed to changes in the cell capacity to produce the proteins.
Figure 6.
DRiP78 regulates CCR5 interaction with G protein subunits during assembly.
a) HEK293 cells were co-transfected with cDNAs encoding DRiP78 WT, HA-CCR5 WT or F/A, and various G protein subunits (Gαi-Rluc, Gβ1-Rluc in presence of Gγ2. Cells were then lysed and immunoprecipitations using an antibody directed against HA were performed. An immunoblot was then performed using an antibody directed against Rluc to reveal the G protein subunits. b) Histogram representation of the results obtained by immunoblotting. c) and d) BRET experiment showing WT CCR5-GFP10 or CCR5 F/A-eGFP interaction with Rluc-tagged Gαi, Gαs and Gβ1 subunits. * = p<0.05; ** = p<0.01 compared with negative controls. Results are representative of 3 independent experiments.
Figure 7.
DRiP78 effect on Jurkat cell migration.
48 hours post-transfection, Jurkat cells overexpressing WT CCR5 or CCRF F/A, and DRiP78 constructs were placed in the upper chamber of a transwell plate and incubated with 10 ng/mL of RANTES for 5 hours. Cells that migrated through the membrane were then counted. Results are representative of 3 independent experiments, and * = p<0.05 compared with controls.