Fig. 4.
Dimeric and monomeric CXCL12 functionally activate CXCR4. (A) The affinities of CXCL12 variants for CXCR4 were determined by 125I-CXCL12 displacement. Kd values for binding of CXCL12WT, CXCL12H25R, and the CXCL122 were calculated as 25, 25, and 150 nM, respectively, from their corresponding logEC50s of -7.59 ± 0.07, -7.62 ± 0.10, and -6.78 ± 0.11. (B) Radioligand displacement of CXCR7-bound 125I-CXCL12 resulted in weaker affinity of CXCL122 (Kd > 1 mM) compared to CXCL12WT (Kd = 28 nM) and CXCL12H25R (Kd = 15 nM). (C) Intracellular calcium mobilization of HCT116 cells stimulated by 10 nM CXCL12 variants was measured in the presence or absence of the Gαi inhibitor, pertussis toxin (PTX). (D) Inhibition of forskolin-induced cAMP production was measured in HEK293 cells cotransfected with CXCR4 and an intramolecular Epac-BRET2 sensor. Measurements were performed 5 min after incubation with CXCL12 variants. (E) Stimulation of HCT116 cells with 10 nM CXCL12WT, (white bar), CXCL12H25R (gray bar), or CXCL122 (black bar) evoked phosphorylation of ERK1/2. Wild-type and monomer resulted in a slower increase in ERK1/2 phosphorylation that was statistically greater than unstimulated (NS) cells starting at 10 min and continuing through 30 min of stimulation. In contrast, dimer resulted in rapid and transient ERK1/2 phosphorylation at 5 min and 10 min and returned to baseline after 15 min and 30 min of treatment. Thirty-minute stimulation with 5 nM epidermal growth factor (EGF) served as a positive control. Data are the ratio of phosphorylated and total ERK1/2 protein normalized to the unstimulated control. Values are mean ± SEM, n = 3. Asterisk indicates statistically significant (P ≤ 0.05) differences. (F) HEK293 cells transiently coexpressing β-arrestin-2-RLuc as a BRET donor and CXCR4-YFP as BRET acceptor were stimulated with the indicated concentrations of CXCL12 variants. Data are the mean ± SD, n = 3. Statistical significance was determined by one-way ANOVA/Dunnett posttest. *, P ≤ 0.01. (G) Time-course measurements of CXCR4 β-arrestin-2 recruitment following stimulation 200 nM CXCL12WT (gray line), CXCL12H25R (dashed line), or CXCL122 (black line). Values are mean ± SD of three separate experiments. (H) Surface CXCR4 levels were measured by flow cytometry after stimulation with CXCL12WT, CXCL12H25R, or CXCL122 for the indicated times (n = 2).