CXCR7 Scavenges CXCL12 Figure 4. Ligand-independent receptor cycling. (A) MDCK cells expressing CXCR7 or CXCR4 were treated on ice with proteinase K to eliminate receptor epitopes from the surface. Cells were washed and incubated at 37uC in medium. At the indicated times aliquots were removed from the incubation and receptor surface expression measured by FACS analysis. (B) Daudi B cells expressing endogenous CXCR7, CXCR4, and transferrin receptor were treated with proteinase K as in (A), and subjected to FACS analysis. (C) MDCK cells expressing membrane-anchored mCherry (red color) and CXCR7 or CXCR4 were incubated with receptor specific monoclonal antibodies at 37uC for 30 min. Cells were fixed, permeabilized and stained with goat anti-mouse F(ab)2 Alexa 488. Red: Actin stained with Alexa Fluor 594 phalloidin; blue Nuclei stained with DAPI. Ligand-independent receptor internalization was visualized by confocal microscopy. Representative data shown from one experiment out of 5. (D) MDCK cells expression CXCR7, CXCR4, or a vector coding for a CXCR7 lacking the cytoplasmic C terminus (DCXCR7) were incubated on ice with receptor-specific antibodies. After removal of unbound antibodies cells were shifted to 37uC. At the indicated times aliquots were withdrawn and incubated with secondary goat anti-mouse F(ab)2 Alexa 488 at 0uC. Receptor surface expression was measured by FACS analysis. doi:10.1371/journal.pone.0009175.g004 receptor cycling MDCK cells expressing CXCR4,CXCR7 or DCXCR7 were incubated with the respective antibodies at 0uC. Excess of antibody was removed and the cells shifted to 37uC in the presence of cycloheximide. Detection of the primary antibodies bound to surface-expressed receptors by FACS analysis revealed a rapid time dependent decrease of CXCR7 (Figure 4D). The loss of primary antibody from the cell surface is in agreement with ligand-independent internalization of CXCR7. Cells express- ing DCXCR7 showed a marginal loss of primary antibody from the cells surface over time, in accordance with the notion that DCXCR7 does not internalize upon ligand exposure [5] and the failure of mediating CXCL12 degradation (Figure 3D). As expected primary antibody bound to CXCR4 expressed on the plasma membrane decreased only marginally consistent with the negligible spontaneous internalization. As both, ligand indepen- dent cycling and CXCL12-induced receptor internalization PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9175