CXCR7 selectively interferes with CXCR4-mediated G protein activation. (A) CXCR7 expression was achieved in parental HEK-293T cells or CXCR4-expressing cells using a lentiviral-based strategy. Cell surface expression levels of CXCR4 (top panel) and CXCR7 (bottom panel) in parental (filled histograms) or cells expressing either (black histograms) or both (light gray histograms) receptors were determined by flow cytometry. (B) One hundred nanomoles of CXCL12-induced ³⁵S-GTP-γS binding to membranes from parental cells or cells expressing CXCR4, CXCR7, or both receptors, in the absence or presence of anti-CXCR4 12G5 or anti-CXCR7 9C4 mAbs at the indicated concentrations. Results are expressed as percentage of the basal binding measured in the absence of ligand. *P < .05, compared with CXCL12-induced binding to CXCR4- and CXCR7-expressing membranes without the anti-CXCR4 12G5 mAb. (C) ³⁵S-GTP-γS binding to membranes from cells expressing either CXCR4 alone or with CXCR7 in the presence of increasing CXCL12 concentrations. Results (mean ± SEM) are representative from 5 independent experiments performed in duplicate. EC₅₀ values were significantly different in the presence of CXCR7 compared with membranes from cells expressing CXCR4 alone (P < .01). (D) CXCR7 expression was achieved in HEK-293T cells coexpressing CD4, CXCR4, and CCR5 using a lentiviral-based strategy. ³⁵S-GTP-γS binding to membranes was measured in response to CXCL12 or the CCR5 agonist CCL4/MIP-1β at the indicated concentrations. Representative results of 3 independent experiments are shown. EC₅₀ values were significantly different in the presence of CXCR7 compared with control conditions with CXCR4 and CCR5 alone (P < .05).