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First published online 12 October 2004
doi: 10.1242/jcs.01398

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The C-terminal domain LLKIL motif of CXCR2 is required for ligand-mediated polarization of early signals during chemotaxis

Department of Veteran Affairs, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

View larger version (28K): [in a new window]   Fig. 1. Recruitment of PH-Akt-GFP to the leading edge of the cell during chemotaxis. A DNA vector expressing PH-Akt-GFP was transiently transfected into HEK293 cells stably expressing either (A) WT CXCR2 or (B) LLKIL mutant CXCR2. Time-lapse fluorescence microscopic images were taken every minute immediately after a point source of ligand (1 µg/ml MIP-2) was applied. The number in each picture shows the time-lapse in minutes and the arrow indicates the direction of ligand application. The arrowheads point towards the accumulation of PH-Akt-GFP. Scale bars, 20 µm. Each experiment was repeated at least three times and representative images are shown. The time-lapse video clips are available online (Movies 1 and 2 in supplementary material).

View larger version (17K): [in a new window]   Fig. 2. Akt activity is required for CXCR2-mediated chemotaxis. HEK293 cells stably expressing WT CXCR2 were transiently transfected with either dominant negative Akt (black bars) or vector control (open bars). The chemotaxis assays were performed in a modified Boyden chamber. Duplicates were used for each sample and the number of cells in ten fields was counted for each well. The chemotaxis index was calculated as described in Materials and Methods. Error bars indicate the s.e.m. ANOVA analysis was performed to evaluate the statistical difference between vector control and dominant negative Akt (P<0.01).

View larger version (57K): [in a new window]   Fig. 3. Inhibition of CXCR2 internalization by dominant negative dynamin results in inhibition of PH-Akt recruitment. HEK 293 cells stably expressing WT CXCR2 were co-transfected with PH-Akt-GFP, and either (A) wild-type dynamin or (B) dominant negative dynamin (K44A). Time-lapsed fluorescence microscopy was carried out for each group of transfected cells. Point source of ligand (MIP-2) was given as described in Materials and Methods and the arrow indicates the direction of ligand application. Scale bars, 20 µm. The number inside each picture gives the time-lapse in minutes after initiation of ligand stimulation.

View larger version (35K): [in a new window]   Fig. 4. FRET images of Rac1 in chemotaxing cells. Membrane-bound Rac1 activities were tested by FRET assay in HEK 293 cells expressing (A) WT or (B) LLKIL mutant CXCR2. FRET images were made as ratio images from two different fluorescent images and plotted with pseudocolor (red, highest activity; blue, lowest activity. Although the overall activities were increased in both types of cells expressing either receptor, the distribution of Rac1 activities was only polarized to the leading pseudopod in WT CXCR2-expressing cell with a higher level in the leading edge. The number in each image indicates the time-lapse in minutes after ligand application, the arrow indicates the direction of ligand application. Scale bars, 20 µm. Each experiment was repeated at least three times and representative images are shown. The time-lapse video clips are available in online (Movies 3 and 4 in supplementary material).

View larger version (26K): [in a new window]   Fig. 5. FRET images of Cdc42 in chemotaxing cells. Membrane-bound Cdc42 activities were tested by FRET assay in HEK 293 cells expressing (A) WT or (B) LLKIL mutant CXCR2. FRET images were made as ratio images from two different fluorescent images and plotted with pseudocolor, in which, red represents the highest activity and blue is lowest. Although the overall activities were increased in both types of cells expressing either receptor, the distribution of Cdc42 activities was only polarized in WT CXCR2-expressing cell with a higher level in the leading edge. The number showing in each image indicates the time-lapse in minutes after ligand application, the arrow indicates the direction of ligand application. Scale bars, 20 µm. Each experiment was repeated at least three times and representative images are shown. The time-lapse video clips are available online (Movies 5 and 6 in supplementary material).

View larger version (27K): [in a new window]   Fig. 6. Activation of Cdc42 and Erk1/2 in HEK293 cells expressing either WT or LLKIL mutant receptors upon agonist stimulation. HEK 293 cells stably expressing either WT CXCR2 or LLKIL mutant receptor were treated without or with MIP-2 (100 ng/ml) for 5 minutes. The affinity precipitation assay (see Materials and Methods for details) was performed for Cdc42 activity and western blots with an p-Erk1/2-specific antibody was performed for the Erk1/2 activation test. Experiments were repeated at least three times with similar results. (A) A representative western blot from three experiments. (B) Quantification of the western blots with error bars for s.e.m. Significant differences of ligand treatment versus non-treatment are indicated by asterisks (P<0.05, Student's t-test).

View larger version (17K): [in a new window]   Fig. 7. The inhibition of dominant negative and constitutively active Cdc42 on chemotaxis in HEK 293 cells. HEK 293 cells stably expressing WT CXCR2 were transfected with pCDNA3 vector (control, black bars) or dominant negative Cdc42 (DN-Cdc42, gray bars) or constitutively active Cdc42 (CA-Cdc42, open bars) constructs. Chemotaxis assays were performed 48 hours after transfection. Each assay was repeated at least three times. Error bars give s.e.m. An ANOVA was performed for the comparison between vector control and either dominant negative or constitutive active Cdc42 (P<0.01).