Early studies revealed that RhoB regulates the transport of the EGF receptor from late endosomes to the lysosome (Mellor et al., 1998
). Because RhoB interacts with a number of proteins that are not isoform-specific, the function of RhoB has been difficult to establish. One possibility is that RhoB regulates signaling events by recruiting effectors to the endosomal compartments. For example, protein kinase C (PKC)-related kinase 1 (PRK1) colocalizes with RhoB on endosomal compartments and expression of the kinase-dead mutant PRK1 antagonizes the effect of RhoB on EGF receptor trafficking (Gampel et al., 1999
; Mellor et al., 1998
). Recent work identified RhoB-positive endosomes as a source of new actin polymerization through the action of Scar1 (WAVE1) in a Src-dependent manner (Sandilands et al., 2004
). We have shown here that RhoB plays a role in the intracellular CXCR2 sorting decision and that interference with RhoB function alters CXCR2-mediated chemotaxis.
The RhoB T19N and Myc-RhoB Q63L mutants and also RhoB siRNA severely impair CXCR2-mediated chemotaxis. Although the membranes were coated with collagen IV for these experiments, it does not appear that differences in invasion contribute significantly to the inhibition. We did not observe any difference in the migration of vector-transduced cells and cells transduced with Myc-RhoB T19N in the absence of CXCL8, suggesting that there is no difference in the ligand-independent invasiveness of these two cell lines. Although, when Myc-RhoB Q63L is expressed, there is a decrease in the number of migrated cells in the absence of CXCL8, this would not account for the significant inhibition of CXCL8-mediated chemotaxis. When either mutant was expressed, we did not observe any significant differences in the activation of MAPK or PI 3-kinase signaling pathways over a 30-minute time course, as measured by phosphorylation of ERK1/2 and Akt, respectively. However, it is possible that localized signaling is affected when these mutants are expressed.
Expression of the dominant-negative (T19N) RhoB mutant results in accumulation of CXCR2 in the perinuclear recycling compartment and in enhanced recycling to the plasma membrane after long-term ligand stimulation (). Also, CXCR2 degradation is impaired when this mutant is expressed. These results suggest that RhoB GTPase activity is required for CXCR2 entry to the lysosome after long-term ligand stimulation. However, this accumulation of CXCR2 in the perinuclear recycling compartment and the failure to enter the lysosome and degrade are not a result of the inability of CXCR2 to exit the recycling compartment and return to the cell surface, because 125I-CXCL8-binding studies reveal that the receptor does return to the plasma membrane and bind ligand. We hypothesize that the accumulation of CXCR2 in the Rab11a compartment is a result of a ‘system overload’ because virtually all CXCR2 in the cells is passing through this compartment to return to the plasma membrane. CXCR2 is probably rapidly re-internalized upon return to the plasma membrane, reducing the time it resides at the plasma membrane. This may contribute to the inability to visualize the receptor at the plasma membrane by microscopy. Moreover, there are no differences in EGFP-Rab11a endosome motility, so differences in the rate of movement of the endosomes cannot account for the failure to enter the lysosome. Thus, it appears that expression of Myc-RhoB T19N impairs the ability of CXCR2 to enter the lysosome and causes prolonged CXCR2 recycling.
Fig. 10 Schematic representation of the effects the T19N and Q63L RhoB mutants have on CXCR2 trafficking. After 30 minutes of CXCL8 stimulation CXCR2 traffics to the Rab11a perinuclear recycling compartment and recycles back to the plasma membrane. CXCR2 traffics (more ...)
Because expression of the RhoB T19N mutant results in decreased degradation and increased recycling of CXCR2, we expected that chemotaxis might be enhanced in cells expressing this mutant. Surprisingly, the opposite effect was observed, and chemotaxis was impaired in cells expressing RhoB T19N. Although CXCR2 is efficiently recycled in these cells, the recycled receptor may not be functional upon its return to the plasma membrane. For example, the dephosphorylation of the receptor is thought to be necessary for subsequent resensitization of CXCR2. Prior studies have shown that, when the CCR5 receptor is stimulated with an N-terminally modified CCL5 ligand, the receptor fails to efficiently respond to subsequent ligand challenge (Mack et al., 1998
; Proudfoot et al., 1999
). Further investigation revealed that, when stimulated with this modified ligand, CCR5 is not dephosphorylated and therefore has only a very brief period of residency at the plasma membrane before being re-internalized (Signoret et al., 2000
). In addition, studies examining the interaction of protein phosphatase 2A (PP2A) and CXCR2 demonstrated that inhibition of PP2A activity by treatment with okadaic acid impairs CXCR2-mediated chemotaxis and Ca2+
mobilization in response to CXCL8 (Fan et al., 2001
). These studies suggest that receptor dephosphorylation may not be required for recycling but for establishing a functional receptor back at the plasma membrane. Interestingly, a novel interaction between RhoB and the catalytic subunit of PP2A was recently identified, further suggesting a potential link between dephosphorylation and RhoB (Lee et al., 2007
). It is not clear where in the endosomal trafficking pathway CXCR2 is dephosphorylated. In the future it will be of interest to examine whether CXCR2 dephosphorylation is affected in cells expressing RhoB T19N.
Treatment of cells with the actin-disrupting drugs cytochalasin D and latrunculin B resulted in a similar accumulation of CXCR2 in the Rab11a compartment as the RhoB T19N mutant. Since RhoB coordinates actin polymerization on endosomes (Fernandez-Borja et al., 2005
; Sandilands et al., 2004
), we suspected that RhoB regulates the motility of the Rab11a-positive endosomes in an actin-dependent manner. To explore this possibility we examined the motility of EGFP-Rab11a-positive endosomes using time-lapse confocal microscopy. The overall velocity of these endosomes and maximum distance traveled did not significantly differ between the vector-transfected cells and cells transfected with RhoB T19N. These results indicate that Rab11a-positive endosomes are able to exit the perinuclear region and return to the plasma membrane when RhoB T19N is expressed.
It is not clear whether the RhoB T19N mutant actually promotes trafficking of CXCR2 to the Rab11a compartment or merely inhibits the ability of the receptor to enter the lysosome for degradation, which results in receptor recycling by default. Moreover, it is unclear whether the RhoB Q63L mutant inhibits entry into the perinuclear recycling compartment or actually promotes accumulation in the sorting endosome and colocalization with Rab4, mannose-6-phosphate receptor and Rab7. Similarly, expression of activated RhoB Q63L enhances localization of rhophilin-2 to late endosomes but not to the lysosome (Steuve et al., 2006
). Studies using the EGF receptor combined with our results suggest a broad role for RhoB in receptor sorting, which is not limited to a specific sorting decision. Alternatively, RhoB may play a variant role in the trafficking of receptors, such as CXCR2, that can be differentially sorted to the recycling endosome or the lysosome compared with those receptors, such as the EGF receptor, that are predominantly sorted to the lysosome. RhoB can recruit proteins that may be involved in intracellular trafficking to endosomal membranes such as Dia1 (Fernandez-Borja et al., 2005
) and PRK1 (Mellor et al., 1998
). The recruitment of different effectors to RhoB may mediate the various responses elicited by RhoB.
Interestingly, the RhoB Q63L mutant impairs the ability of CXCR2 to enter the perinuclear recycling compartment but the receptor is still able to recycle back to the plasma membrane (). The degradation of CXCR2 and colocalization of CXCR2 with lysosomal markers is also impaired when the Q63L mutant is expressed. This finding implicates not only the ability of RhoB to exchange GDP for GTP but also the ability of RhoB to hydrolyze GTP for proper function. It appears that GDP-bound RhoB specifies sorting to the Rab11a perinuclear recycling compartment, whereas GTP-bound RhoB and subsequent GTP hydrolysis is necessary for lysosomal sorting.
The colocalization of CXCR2 with Rab4 and the mannose-6-phosphate receptor indicate that CXCR2 enters the sorting compartment, and recycles back to the plasma membrane through alternative recycling pathways. It is interesting that CXCR2 may enter the trans-Golgi network and recycle back to the plasma membrane through this compartment. Our data indicate that CXCR2 recycling is a default pathway, whereas degradation is largely mediated through the activation of CXCL8 or RhoB.
In summary, the dominant-negative (T19N) RhoB mutant, the GTPase-deficient activated (Q63L) RhoB mutant and siRNA directed against RhoB all impair CXCR2-mediated chemotaxis. This impairment is accompanied by the failure of CXCR2 to traffic appropriately. Normal activity of RhoB is essential for CXCR2 degradation in the lysosome and recycling through the perinuclear Rab11a-positive compartment. We therefore propose that RhoB plays a role in the CXCR2 sorting decision. The ability of the cell to differentially sort chemokine receptors is crucial for the chemotactic response. These results establish for the first time that RhoB plays a role in the differential sorting of a chemokine receptor.