FIGURE 2.

CXCR4·CXCR7 complex constitutively recruits β-arrestin. A, HEK293 cells were co-transfected with CXCR7-FLAG and β-arrestin-eGFP, along with either CXCR4 or CCR5, and treated with 100 nm SDF-1 for the indicated time. Lysates were immunoprecipitated (IP) with polyclonal anti-FLAG antibodies and subjected to Western immunoblot (IB) analysis using anti-GFP (upper panel) and anti-FLAG (middle panel) antibodies, respectively. Increased recruitment of β-arrestin to membranes containing CXCR7 is observed at base line in the presence of CXCR4 but not CCR5 (upper panel). The same trend is observed after 5 and 30 min of stimulation with SDF-1. The lower panel shows the input levels of β-arrestin-eGFP expression using anti-GFP antibody. B, quantification of β-arrestin recruitment kinetics in membranes containing CXCR7 alone or when CXCR7 is co-expressed with CXCR4 or CCR5. Data represent mean ± S.E. (n = 3). A.U., arbitrary units. C, HEK293 cells were co-transfected with CXCR7-FLAG and β-arrestin-eGFP, along with either control vector or CXCR4-C9, and treated with 100 nm SDF-1 or ITAC for 30 min. Lysates were immunoprecipitated with polyclonal anti-FLAG antibody and subjected to Western immunoblot analysis using anti-GFP antibody (bottom panel). Quantification of β-arrestin-recruitment is depicted in the top panel. Data represent results from at least three independent experiments and are expressed as mean ± S.E. The maximum response from β-arrestin recruitment to CXCR4-CXCR7 membranes is taken as 100%. Stimulation with ITAC does not result in increased recruitment of β-arrestin to the CXCR4-CXCR7-expressing membranes. D, immunofluorescence image of Neuro2A cell co-expressing CXCR4, CXCR7, and βArr-eGFP. A shows the merged image of CXCR4/CXCR7/βArr-eGFP. B–D show a higher magnification of a small area (white square, A) demonstrating co-localization of all these partners.