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CXCR7 controls competition for recruitment of β-arrestin 2 in cells expressing both CXCR4 and CXCR7.

Coggins NL, Trakimas D, Chang SL, Ehrlich A, Ray P, Luker KE, Linderman JJ, Luker GD - PLoS ONE (2014)

Bottom Line: We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4.These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7.These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Imaging, Department of Radiology, Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the "competition" with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

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Kinetics of β-arrestin 2 recruitment to CXCR4 or CXCR7.(A and B) MDA-MB-231 breast cancer cells expressing CXCR4-CBRN/β-arrestin 2-CBC (A) or CXCR7-CBRN/β-arrestin 2-CBC (B) were treated with increasing concentrations of CXCL12-α (ng/mL) as denoted in the legend. Data were collected as photon flux units. Photon flux values for each time point then were normalized to values obtained for control cells not incubated with CXCL12 at each time point through 40 min and at 90 min. Data are expressed as mean values ± SEM for fold change relative to control (n = 4 per point). (C and D) Experimental data were used to tune parameters for a computational model describing numbers of receptors per cell bound to β-arrestin 2. Model outputs for CXCR4 (C) and CXCR7 (D) were plotted as fold change relative to cells not treated with CXCL12. (E, F) Internalization of cell surface CXCR4 (E) or CXCR7 (F) following 40 min or 30 min, respectively, of incubation with CXCL12 was measured by flow cytometry. Values for 0 ng/ml CXCL12 describe internalization of CXCR4 or CXCR7 in the absence of ligand. Experimental data for CXCR7 were replotted based on previously published results [12]. Model fits also are shown.
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pone-0098328-g003: Kinetics of β-arrestin 2 recruitment to CXCR4 or CXCR7.(A and B) MDA-MB-231 breast cancer cells expressing CXCR4-CBRN/β-arrestin 2-CBC (A) or CXCR7-CBRN/β-arrestin 2-CBC (B) were treated with increasing concentrations of CXCL12-α (ng/mL) as denoted in the legend. Data were collected as photon flux units. Photon flux values for each time point then were normalized to values obtained for control cells not incubated with CXCL12 at each time point through 40 min and at 90 min. Data are expressed as mean values ± SEM for fold change relative to control (n = 4 per point). (C and D) Experimental data were used to tune parameters for a computational model describing numbers of receptors per cell bound to β-arrestin 2. Model outputs for CXCR4 (C) and CXCR7 (D) were plotted as fold change relative to cells not treated with CXCL12. (E, F) Internalization of cell surface CXCR4 (E) or CXCR7 (F) following 40 min or 30 min, respectively, of incubation with CXCL12 was measured by flow cytometry. Values for 0 ng/ml CXCL12 describe internalization of CXCR4 or CXCR7 in the absence of ligand. Experimental data for CXCR7 were replotted based on previously published results [12]. Model fits also are shown.

Mentions: Association of CXCR4-CBRN with β-arrestin 2-CBC increased rapidly in a concentration-dependent manner after adding CXCL12 (Fig. 3A, Fig. A in File S1). As little as 37 ng/mL CXCL12 increased association of CXCR4 and β-arrestin 2 above basal levels, and 1000 ng/mL produced a 4-fold increase in bioluminescence. Interaction of CXCR4 and β-arrestin 2 peaked at ≈20–22 minutes for cells treated with 1000 ng/mL CXCL12, while plateau levels occurred slightly later for cells incubated with lower concentrations of CXCL12. Association of CXCR4-CBRN with β-arrestin 2-CBC decreased in a concentration-dependent manner by 90 min.


CXCR7 controls competition for recruitment of β-arrestin 2 in cells expressing both CXCR4 and CXCR7.

Coggins NL, Trakimas D, Chang SL, Ehrlich A, Ray P, Luker KE, Linderman JJ, Luker GD - PLoS ONE (2014)

Kinetics of β-arrestin 2 recruitment to CXCR4 or CXCR7.(A and B) MDA-MB-231 breast cancer cells expressing CXCR4-CBRN/β-arrestin 2-CBC (A) or CXCR7-CBRN/β-arrestin 2-CBC (B) were treated with increasing concentrations of CXCL12-α (ng/mL) as denoted in the legend. Data were collected as photon flux units. Photon flux values for each time point then were normalized to values obtained for control cells not incubated with CXCL12 at each time point through 40 min and at 90 min. Data are expressed as mean values ± SEM for fold change relative to control (n = 4 per point). (C and D) Experimental data were used to tune parameters for a computational model describing numbers of receptors per cell bound to β-arrestin 2. Model outputs for CXCR4 (C) and CXCR7 (D) were plotted as fold change relative to cells not treated with CXCL12. (E, F) Internalization of cell surface CXCR4 (E) or CXCR7 (F) following 40 min or 30 min, respectively, of incubation with CXCL12 was measured by flow cytometry. Values for 0 ng/ml CXCL12 describe internalization of CXCR4 or CXCR7 in the absence of ligand. Experimental data for CXCR7 were replotted based on previously published results [12]. Model fits also are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4045718&req=5

pone-0098328-g003: Kinetics of β-arrestin 2 recruitment to CXCR4 or CXCR7.(A and B) MDA-MB-231 breast cancer cells expressing CXCR4-CBRN/β-arrestin 2-CBC (A) or CXCR7-CBRN/β-arrestin 2-CBC (B) were treated with increasing concentrations of CXCL12-α (ng/mL) as denoted in the legend. Data were collected as photon flux units. Photon flux values for each time point then were normalized to values obtained for control cells not incubated with CXCL12 at each time point through 40 min and at 90 min. Data are expressed as mean values ± SEM for fold change relative to control (n = 4 per point). (C and D) Experimental data were used to tune parameters for a computational model describing numbers of receptors per cell bound to β-arrestin 2. Model outputs for CXCR4 (C) and CXCR7 (D) were plotted as fold change relative to cells not treated with CXCL12. (E, F) Internalization of cell surface CXCR4 (E) or CXCR7 (F) following 40 min or 30 min, respectively, of incubation with CXCL12 was measured by flow cytometry. Values for 0 ng/ml CXCL12 describe internalization of CXCR4 or CXCR7 in the absence of ligand. Experimental data for CXCR7 were replotted based on previously published results [12]. Model fits also are shown.
Mentions: Association of CXCR4-CBRN with β-arrestin 2-CBC increased rapidly in a concentration-dependent manner after adding CXCL12 (Fig. 3A, Fig. A in File S1). As little as 37 ng/mL CXCL12 increased association of CXCR4 and β-arrestin 2 above basal levels, and 1000 ng/mL produced a 4-fold increase in bioluminescence. Interaction of CXCR4 and β-arrestin 2 peaked at ≈20–22 minutes for cells treated with 1000 ng/mL CXCL12, while plateau levels occurred slightly later for cells incubated with lower concentrations of CXCL12. Association of CXCR4-CBRN with β-arrestin 2-CBC decreased in a concentration-dependent manner by 90 min.

Bottom Line: We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4.These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7.These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Imaging, Department of Radiology, Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the "competition" with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

Show MeSH
Related in: MedlinePlus