Leukocyte analysis from WHIM syndrome patients reveals a pivotal role for GRK3 in CXCR4 signaling
Karl Balabanian, … , Fernando Arenzana-Seisdedos, Françoise Bachelerie
Karl Balabanian, … , Fernando Arenzana-Seisdedos, Françoise Bachelerie
Published February 14, 2008
Citation Information: J Clin Invest. 2008;118(3):1074-1084. https://doi.org/10.1172/JCI33187.
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Research Article Immunology

Leukocyte analysis from WHIM syndrome patients reveals a pivotal role for GRK3 in CXCR4 signaling

Abstract

Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF (WHIMWT) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did WHIMWT cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIMWT leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIMWT patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related WHIMWT disorder.

Authors

Karl Balabanian, Angélique Levoye, Lysiane Klemm, Bernard Lagane, Olivier Hermine, Julie Harriague, Françoise Baleux, Fernando Arenzana-Seisdedos, Françoise Bachelerie

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Figure 4

GRK3-mediated internalization and desensitization of CXCR4.

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GRK3-mediated internalization and desensitization of CXCR4. (A) Aforemen...
(A) Aforementioned fibroblasts from CTRL#1, P3, and P4 subjects expressing CXCR4 were nucleoporated with 5 μg of either pcDNA1 (vector) or GRK2, -3, -5, or -6 construct and treated 15 h after transfection with 200 nM CXCL12. We always controlled the efficiency of GRK overexpression by IB (Supplemental Figure 2C). (B) CXCL12-promoted internalization of CXCR4WT or CXCR41013 in fibroblasts from CTRL#1 and P3 nucleoporated with 5 μg of either pcDNA1 (vector) or plasmid encoding GRK3 cDNA. (C) Expression of GRK products in fibroblasts from healthy subjects nucleoporated with 5 μg SCR, GRK2, or GRK3 siRNAs. Three days after transfection, GRK mRNA levels were assessed by quantitative PCR (left and middle panels) and normalized to those of IDUA. IB of proteins from whole-cell lysates either with an anti-GRK2/3 mAb (data not shown) or an anti-GRK2 or anti-GRK3 Ab (right panel) revealed proteins of expected molecular mass (~80 kDa). GRK2 or -3 siRNAs had no effect on expression levels of endogenous GRK6 or β-arrestin2 (molecular mass ~65 and ~46 kDa, respectively), as detected using selective Abs (Supplemental Figures 1 and 2). (D) Cell surface expression levels of CXCR4 in GRK2 or -3 siRNA-transfected fibroblasts treated with CXCL12. GRK2 or -3 siRNAs had no effect on expression levels of CXCR4 at the membrane of unstimulated cells (data not shown). (E) CXCR4-transduced fibroblasts from CTRL#1, P3, and P4 individuals were nucleoporated with 5 μg of pcDNA1 (vector) or GRK2 or -3 construct and tested for CXCL12-triggered actin polymerization. Results are means ± SD of 3–6 independent experiments (A and B; C, left and middle panels; and D and E) or are representative of >5 independent determinations (C, right panel). *P < 0.05; #P < 0.005 compared with fibroblasts transfected with vector (A and B) or SCR siRNAs (C and D).