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 3

β-arrestin–mediated internalization of CXCR4.

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β-arrestin–mediated internalization of CXCR4. (A and B) CXCR4-transduced...
(A and B) CXCR4-transduced fibroblasts from healthy, P3, and P4 subjects were nucleoporated with 5 μg pN1-EGFP (vector), pβ-arrestin1–EGFP (β-arr1), or pβ-arrestin2–EGFP (β-arr2) and treated 15 h after transfection with 200 nM CXCL12 (left panels). Transfection rates >60% were obtained in all cell types as evaluated by flow cytometry. Results are expressed as percentage of CXCR4 expression (100% corresponding to CXCR4 expression at the surface of GFP+-gated cells incubated in medium alone). Probing of protein extracts with an anti–β-arrestin1 or –β-arrestin2 Ab revealed proteins of expected molecular mass (~47 and ~46 kDa for β-arrestin1 and -2, respectively) in each sample (right panels). LDH (~35 kDa) was used as a loading control. (C) Membrane expression levels of CXCR4WT or CXCR41013 upon CXCL12 stimulation in CTRL#1 fibroblasts nucleoporated with 5 μg pN1-EGFP (vector) or pβ-arrestin2–EGFP. After transduction, CXCR4WT and CXCR41013 receptors were expressed at similar levels at the surface of unstimulated cells (data not shown). Results are means ± SD of >3 independent experiments (A–C) or are representative out of >3 independent determinations (A and B, right panels). *P < 0.05; **P < 0.005 compared with fibroblasts transfected with vector.