Functional characterization of HA-CXCR4-S346-8A and HA-CXCR4-E343K mutants.

<p><b>A–M</b>, Wildtype CXCR4 (X4-WT) and the mutants were transiently transfected in HEK293 cells. <b>A</b>, Surface receptor levels of the transfectants were analyzed by ELISA using anti-HA antibody. Results are given as percent of wildtype (WT) levels and represent mean+SEM from two independent experiments with 3 to 5 repeats. <b>B</b>, Homologous radioligand competition binding using ¹²⁵I-CXCL12 (25 pM) and increasing concentrations of unlabeled CXCL12. The three CXCR4 variants have comparable CXCL12 binding properties (WT, 37 nM; E343K and S346-8A, 18 nM). <b>C–H</b>, Surface receptors were pulse-labeled with anti-HA antibody applied to live cells at 4°C. Confocal images show the subcellular localization of labeled receptors after a 30 min interval at 37°C in absence of ligand (C–E) or in the presence of CXCL12 (F–H). Cells transfected with wildtype CXCR4 show major internalization after CXCL12 stimulation (F). In cells transfected with mutant receptors, the majority of the staining remains plasma-membrane associated even after 30 min CXCL12 stimulation (G,H). <b>I</b>, Internalization kinetics of surface receptors labeled with ¹²⁵I-CXCL12. Transfected cells were loaded with ¹²⁵I-CXCL12 at 4°C (pulse), washed, and lysed immediately (starting value) or incubated for the indicated time intervals at 37°C and lysed after an acidic wash (chase). Amounts of internalized radioligand are given as percent of the starting value. Results represent mean ± SEM from two independent experiments with 4 repeats each. Internalization of radioligand-labeled mutant receptors is significantly reduced. *, #: p<0.05; 2way ANOVA vs. wildtype CXCR4. <b>J–M</b>, G protein coupling of wildtype CXCR4 and of the mutants. CXCR4 constructs were co-transfected with the 16z44 chimeric G protein and changes in Ca²⁺_i were monitored. <b>J</b>,<b>K</b>, Single stimulation with 80 nM CXCL12 reveals an increased peak height in cells expressing the mutant CXCR4 receptors. Data were normalized to mean of the wildtype maximum and plotted as averaged traces with SEM. <b>K</b>, Quantitative analysis of the average peak height. *: p<0.05; Student´s t tests vs. wildtype CXCR4. <b>L</b>,<b>M</b>, Averaged traces with SEM showing two subsequent stimulations with CXCL12, using 5 nM and 80 nM concentrations. <b>L</b>, Data was normalized to the mean of the first maximum of wildtype CXCR4. <b>M</b>, For analysis of homologous desensitization, the second peak was expressed as percent of the first peak for each trace before mean values+SEM of the peak ratios were calculated. Increased ratios in the mutants indicate reduced desensitization. *: p<0.05; Student´s t test vs. wildtype CXCR4.