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. 2013 Jun 18;52(24):4184-92.
doi: 10.1021/bi400254f. Epub 2013 Jun 7.

Modulation of the CXC chemokine receptor 4 agonist activity of ubiquitin through C-terminal protein modification

Abhishek Tripathi  1 , Vikas Saini, Adriano Marchese, Brian F Volkman, Wei-Jen Tang, Matthias Majetschak
Affiliations

Modulation of the CXC chemokine receptor 4 agonist activity of ubiquitin through C-terminal protein modification

Abhishek Tripathi et al. Biochemistry. .

Abstract

Extracellular ubiquitin has recently been described as a CXC chemokine receptor (CXCR) 4 agonist. Studies on the structure-function relationship suggested that the C-terminus of ubiquitin facilitates CXCR4 activation. It remains unknown, however, whether C-terminal processing of ubiquitin could be biologically relevant and whether modifications of the ubiquitin C-terminus can modulate CXCR4 activation. We show that C-terminal truncated ubiquitin antagonizes ubiquitin and stromal cell-derived factor (SDF)-1α induced effects on cell signaling and function. Reduction of cell surface expression of insulin degrading enzyme (IDE), which cleaves the C-terminal di-Gly of ubiquitin, enhances ubiquitin induced reduction of cAMP levels in BV2 and THP-1 cells, but does not influence changes in cAMP levels in response to SDF-1α. Reduction of cell surface IDE expression in THP-1 cells also increases the chemotactic activity of ubiquitin. As compared with native ubiquitin, C-terminal Tyr extension of ubiquitin results in reduced CXCR4 mediated effects on cellular cAMP levels and abolishes chemotactic activity. Replacement of C-terminal di-Gly of ubiquitin with di-Val or di-Arg enhances CXCR4 mediated effects on cAMP levels and the di-Arg substitution exerts increased chemotactic activity, when compared with wild type ubiquitin. The chemotactic activities of the di-Val and di-Arg mutants and their effects on cAMP levels can be antagonized with C-terminal truncated ubiquitin. These data suggest that the development of CXCR4 ligands with enhanced agonist activities is possible and that C-terminal processing of ubiquitin could constitute a biological mechanism, which regulates termination of receptor signaling.

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Figures

Figure 1
Figure 1
C-Terminal truncated ubiquitin functions as a CXCR4 antagonist. (A) Forskolin-stimulated THP-1 cells were incubated with wild type ubiquitin, SDF-1α, or C-terminal truncated ubiquitin (Ub(1–74)) and cAMP levels were measured (n = 3). Data are expressed as % of untreated cells (= 100%). (B) Dose-dependent migration of THP-1 cells toward native ubiquitin, SDF-1α, and Ub(1–74), n = 4–5. (C) Dose-dependent migration of THP-1 cells toward 10 nM native ubiquitin or SDF-1α in the absence (−) or presence (+) of 1 µM Ub(1–74), n = 4–5. (D) Forskolin-stimulated THP-1 cells were incubated with ubiquitin, SDF-1α, and Ub(1–74) as indicated and cAMP levels were measured. Data are expressed as % of untreated cells (= 100%), n = 4–8.
Figure 2
Figure 2
Cell surface expression of insulin degrading enzyme (IDE) modulates ubiquitin-induced effects on cell signaling and function. (A) Quantification of IDE cell surface expression by flow cytometry. Thick lines: Cells labeled with rabbit anti-IDE/antirabbit FITC goat IgG. Thin lines: Control; cells labeled with rabbit IgG/antirabbit FITC goat IgG. Gray: Unstained cells. Red: BV-2 cells. Blue: BV-2 shRNA IDE cells. (B) FITC-ubiquitin binding (1 min, 4 °C) to BV-2 (●) and BV-2 shRNA IDE (○) cells. ●: nonspecific binding (NSB) - BV-2 cells. ○: nonspecific binding (NSB) - BV-2 shRNA cells. RFU: relative fluorescence units. (C) Forskolin stimulated BV-2 (gray bars) and BV-2 shRNA cells (open bars) were incubated with ubiquitin and cAMP levels were measured (n = 6). *: p < 0.05 vs untreated BV-2 cells. Data are expressed as % of untreated BV-2 cells (= 100%). (D) Forskolin-stimulated BV-2 (gray bars) and BV-2 shRNA cells (open bars) were incubated with SDF-1α and cAMP levels were measured (n = 6). *: p < 0.05 vs untreated BV-2 cells. Data are expressed as % of untreated BV-2 cells (= 100%). (E) Quantification of IDE cell surface expression on THP-1 cells by flow cytometry after transfection with IDE siRNA (blue) or nontargeting siRNA (red). Thick lines: Cells labeled with rabbit anti-IDE/antirabbit FITC goat IgG. Thin lines: Control; cells labeled with rabbit IgG/antirabbit FITC goat IgG. Gray: Unstained cells. (F) Forskolin-stimulated THP-1 cells after transfection with nontargeting siRNA (ctrl.; gray bars) and IDE siRNA (open bars) were incubated with ubiquitin and cAMP levels were measured (n = 4). *: p < 0.05 vs untreated BV-2 cells. Data are expressed as % of untreated BV-2 cells (= 100%). (G) Migration of THP-1 cells toward ubiquitin (10 nM). Gray bar: Cells after transfection with nontargeting siRNA (ctrl.). Open bar: Cells after transfection with IDE siRNA. *: p < 0.05 vs ctrl.
Figure 3
Figure 3
The ubiquitin C-terminus regulates signaling upon CXCR4 activation. (A) Ribbon diagram of ubiquitin. Gly-75 and Gly-76 are highlighted in magenta. The generated mutations of the C-terminus are shown. (B, C) Forskolin-stimulated THP-1 cells were incubated with the ubiquitin mutants and cAMP levels were measured (n = 3–5). Data are expressed as % of untreated cells (= 100%). The dashed lines show the dose–response curve for wild type ubiquitin from Figure 1A, which were measured in parallel experiments. (B) Open triangles: Ub(1–74)DD; gray solid triangles: Ub(1–74)NN. (C) ● Ub(1–74)RR; gray solid diamonds: Ub(1–74)VV; gray solid squares: Ub(1–74)GGY. (D) Forskoli-stimulated THP-1 cells were incubated with wild type ubiquitin, SDF-1α, or the ubiquitin mutants (10 nM each) in the absence (−) or presence (+) of AMD3100 (10 µM) as in B–D and cAMP levels were measured (n = 3). Data are expressed as % of untreated cells (= 100%). (E) Forskolin-stimulated THP-1 cells were incubated with Ub(1–74)RR or Ub(1–74)VV (10 nM each) in the absence (−) or presence (+) of Ub(1–74) (1 µM) and cAMP levels were measured (n = 3). Data are expressed as % of untreated cells (= 100%).
Figure 4
Figure 4
The ubiquitin C-terminus regulates cell function upon CXCR4 activation. (A) Dose-dependent migration of THP-1 cells toward native ubiquitin (Ub) and Ub(1–74)DD, n = 7. (B) Dose-dependent migration of THP-1 cells toward Ub(1–74)NN and Ub(1–74)VV, n = 7. (C) Dose-dependent migration of THP-1 cells toward Ub(1–74)RR and Ub(1–74)GGY, n = 7. (D) Migration of THP-1 cells toward ubiquitin and the ubiquitin mutants (10 nM each) in the absence (−) or presence (+) of AMD3100 (10 µM), n = 5. (E) Migration of THP-1 cells toward Ub(1–74)RR and Ub(1–74)VV (10 nM each) in the absence (−) or presence (+) of Ub(1–74) (1 µM), n = 3.
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