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Abstract
The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.
Highlights
Heart and brain, leukocyte chemotaxis, and stem cell homing [1,2,3]
To elucidate the mechanisms mediating CXCR4 degradation and ubiquitination, we initially examined the effect of siRNAmediated depletion of arrestin-2 and arrestin-3 on agonist promoted degradation of endogenous CXCR4 in HeLa cells
Inhibition of CXCR4 degradation was more pronounced in cells depleted of arrestin-2 compared with arrestin-3, even though the siRNA-mediated depletion of arrestin-3 was consistently greater than depletion of arrestin-2, suggesting a more important role for arrestin-2 in degradation of activated CXCR4
Summary
Cell Lines, cDNA Constructs, Antibodies, and siRNA— HEK293 (Microbix, Toronto, Canada) and HeLa cells (American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc., Herndon, VA) supplemented with fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). Detection of Ubiquitinated CXCR4—HEK293 cells grown on 10-cm dishes were transfected with arrestin-2, arrestin-3, arrestin-2/3, and control siRNA as described above, followed by co-transfection of HA-CXCR4 and FLAG-ubiquitin the day. Measuring Receptor Internalization—HEK293 cells grown on 10-cm dishes were transfected with siRNA for arrestin-2 and control siRNA as described above, followed by co-transfection of HA-CXCR4 the day. An anti-AIP4 monoclonal antibody (G11; Santa Cruz) was used to immunoprecipitate endogenously expressed AIP4 in HEK293 cells, essentially as previously described [7], followed by SDS-PAGE and immunoblotting to detect endogenous arrestins. For AIP4 and arrestin-2 co-localization experiments, HEK293 cells transiently cotransfected with HA-CXCR4, mCh-AIP4, and YFP-arrestin-2 were serum-starved for 14 –16 h in DMEM containing 0.5% FBS and treated with vehicle or 30 nM CXCL12 for 30 min followed by permeabilization and fixation. Statistical Analysis—The data were analyzed and subjected to statistical analyses using Prism 4.0 software (GraphPad Software, Inc.)
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