Date of Award

2012

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Molecular Biology

Abstract

Ubiquitin has previously been identified as another natural agonist of CXC chemokine receptor 4 (CXCR4). In addition, recent evidence suggests that ubiquitin may activate CXCR4 through a binding site on the receptor, which is distinct from the binding site for the cognate ligand stromal cell-derived factor (SDF)-1α. The cellular consequences of ubiquitin induced CXCR4 activation, however, are still poorly defined and a side-by-side comparison of CXCR4 mediated functions after activation with SDF-1α and ubiquitin is lacking. Such information will be instrumental to better understand the physiological function of CXCR4 and to further define its role as a therapeutic target in various disease processes. Accordingly, the aim of this study was to determine and compare CXCR4 mediated effects on important signal transduction pathways and chemotaxis, a key function of CXCR4, upon receptor activation with ubiquitin and SDF-1α. Utilizing a MAPK array in combination with Western blot experiments, it is shown that activation of CXCR4 with ubiquitin and SDF-1α in THP-1 cells leads to increased phosphorylation of extracellular signal-related kinase (ERK) 1/2, ribosomal S6 kinase (RSK) 1, and protein kinase B (Akt). Analyses of the time progression of the MAPK phosphorylation revealed that both ligands induced a comparable degree of MAPK phosphorylation, which occurred transiently after activation of CXCR4 with ubiquitin and was sustained for at least 30 minutes with SDF-1α. To assess CXCR4 mediated chemotaxis, a filter migration assay was established and optimized. It is shown that THP-1 cells and human peripheral blood mononuclear cells migrate dose dependently towards ubiquitin and SDF-1α. Under optimized conditions, ubiquitin was 4-5 times less efficacious than SDF-1α in promoting chemotaxis, but of similar potency. Pharmacological inhibition of signal transduction molecules that are known to be involved in the regulation of CXCR4 mediated chemotaxis resulted in similar effects on ubiquitin and SDF-1α induced chemotaxis in THP1 cells and PBMCs. This suggests that both ligands rely on a similar pattern of intracellular signaling to induce chemotaxis. In conclusion, activation of CXCR4 with ubiquitin and SDF-1α results in similar intracellular signaling events and functional consequences. With activation of CXCR4 by ubiquitin, however, serine/threonine protein kinase phosphorylations occurred more transiently, which could account for its weaker chemotactic activity, as compared with SDF-1α. Thus, ubiquitin appears to be a weaker CXCR4 agonist than SDF-1α, which may correspond to differential CXCR4 signaling mediated via distinct ligand binding sites on the receptor

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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