Figure 7. V247N mutant constitutively activates Gα_i.

(A and B) Surface expression of CXCR1 WT and V247N on COS-7 cells (A) and TSA-201 cells (B). Cells were incubated with mouse anti human CD128a (CXCR1) antibody at 4°C overnight. After washing three times, the cells were incubated with the secondary antibody (DyLight 549-conjugated goat anti-mouse IgG(H+L)) at RT for 1 hr and counterstained with Dapi. Expression of CXCR1 or the mutants was observed using a confocal microscope. The pink color represents the surface expression of CXCR1 WT or mutant with Dapi shown in blue. (C) Inhibition of forskolin-stimulated cAMP production in COS-7 cells. COS-7 cells were transfected with CXCR1 WT or V247N mutant, as well as Gα_i2, Gβ₁, and Gγ₂ expression vector. IL-8 significantly inhibited forskolin-induced stimulation of cAMP formation in transfected cells whereas V247N mutation inhibited forskolin-induced cAMP levels even in the absence of IL-8. Transfected cells were used for determination of cAMP levels in the absence (open bars) or in the presence of IL-8 (closed bars). The results shown are representative. (D) Inhibition of hCG-stimulated cAMP accumulation by V247N mutant. TSA-201 cells were transfected with CXCR1 WT or V247N mutant, as well as LH receptor expression vector. The TSA-201 cells were pretreated with 0.5 mM IBMX for 30 min followed by treatment with hCG (10⁻⁷ M) in the presence or absence of IL-8 (40 nM) for another 30 min. Following incubation with hCG in the presence or absence of IL-8, cells were lysed and intracellular cAMP measured using a RIA kit as described in “Experimental Procedures”. Data presented are the mean ± SEM for three assays with each condition performed in triplicate.