FIGURE 1.

CXCR7 alters SDF-1 signaling by forming a complex with CXCR4. A, CXCR4 and CXCR7 co-immunoprecipitate when co-expressed in HEK293 cells. Detergent-soluble lysates from HEK293 cells transfected with CXCR4-C9 and CXCR7-FLAG, as indicated, were treated with anti-FLAG antibody to immunoprecipitate (IP) CXCR7-FLAG. The pulldown was subjected to SDS-PAGE followed by Western immunoblot (IB) analysis using 1D4 mAb (upper panel). 1D4 and anti-FLAG antibodies were used to monitor CXCR4-C9 (middle panel) and CXCR7-FLAG expression levels (lower panel), respectively. B, same experiment as in A except transfected cells were preincubated with 11G8, a CXCR7-specific antibody sensitive to receptor conformation, prior to lysis. Co-IP CXCR4-C9 was detected using 1D4 antibody (upper panel), and CXCR7 levels were monitored using ant-FLAG antibody (lower panel). C, immunofluorescent staining of CXCR4 and CXCR7 expressing Neuro2A cells demonstrates co-localization of CXCR4 and CXCR7 at the membrane. D, pCRE-SEAP assay showing inhibition of FK-induced cAMP production by CXCR4 and CXCR7. SDF-1 stimulation inhibits FK-induced cellular cAMP production through CXCR4 but not through CXCR7. Co-expression of CXCR7 along with CXCR4 results in a decrease in FK-induced cellular cAMP production through CXCR4 in a dose-dependent manner. CXCR4 alone, EC₅₀ = 3.4 ± 0.8; CXCR4 ± CXCR7 1:1.5, EC₅₀ = 60 ± 11. E, unlike CXCR7, co-expression of CCR5 with CXCR4 does not lead to alteration in SDF-1-stimulated signaling in the pCRE-SEAP assay. CXCR4 alone, EC₅₀ = 3.4 ± 0.8; CXCR4 ± CCR5 1:1.5, EC₅₀ = 3.1 ± 0.5. F, pCRE-SEAP assay shows recovery of SDF-1 signaling when CXCR4-CXCR7 co-expressing cells were preincubated with 500 nm of ITAC. Data are expressed as mean ± S.E. (n = 3). CXCR4 alone, EC₅₀ = 3.4 ± 0.8; CXCR4 ± CXCR7 1:1.5, EC₅₀ = 60 ± 11; CXCR4 + CXCR7/ITAC, EC₅₀ = 5.5 ± 1.7.