The C-tail and the SHSK motif of CXCR4 regulate activation of G proteins and receptor down-modulation. (A,B) CXCR4^wt, CXCR4¹⁰¹³, CXCR4^wt/Δi3, and CXCR4^1013/Δi3 were transiently expressed in HEK cells. Flow cytometric analysis using PE-conjugated 12G5 confirmed similar expression levels of receptors at the cell surface. Staining of parental cells was used as a negative control (filled peaks). (C) CXCL12-induced [³⁵S]GTPγS binding to membranes from these cells transiently expressing CXCR4^wt (■), CXCR4¹⁰¹³ (□), CXCR4^wt/Δi3 (●), or CXCR4^1013/Δi3 (○) is shown. Membranes were incubated in assay buffer containing 0.1 nM [³⁵S]GTPγS and the indicated concentrations of CXCL12. The data, which are representative of 4 independent experiments performed in triplicate, are expressed as a percentage of the basal binding to CXCR4^wt-expressing membranes. (D) Receptor expression levels following stimulation with 200 nM CXCL12 for 45 minutes at 37°C are shown. Results (means ± SD; n = 3) indicate the amount of receptor that remains at the surface of HEK cells expressing CXCR4^wt (□), CXCR4¹⁰¹³ (), CXCR4^wt/Δi3 (■), or CXCR4^{1013/ Δi3} (▧) after incubation with CXCL12 compared with untreated cells (n.s.; ).