Date of Award
Master of Science (MS)
Pharmacology and Experimental Therapeutics
CXCR4 is a chemokine receptor that is overexpressed in multiple disease states, including cancer. Understanding the mechanisms by which cells regulate CXCR4 expression is of high importance, as they can reveal downstream effectors that can potentially be targeted pharmacologically to more effectively treat diseases with fewer side effects.
CXCR4 is internalized in response to stimulation by its ligand CXCL12, and localizes to early endosomes as part of a homologous desensitization mechanism. From the endosome, CXCR4 can enter one of two pathways whereby it is either recycled back to the plasma membrane, where it can undergo another signaling event, or it is targeted for lysosomal degradation via the ESCRT pathway in a ubiquitin-dependent fashion. It is known that an interaction between the proteins β-arrestin-1 and STAM-1 (a subunit of ESCRT-0) on endosomal membranes plays a key role in sorting CXCR4 to the degradative pathway, and that disrupting this interaction can accelerate CXCR4 degradation. Therefore, the β-arrestin-1/STAM-1 complex represents a potential target by which to modulate cellular CXCR4 levels.
The goal of this project was to develop an assay that can monitor the interaction between β-arrestin-1 and STAM-1 in live cells, which can be used to study their binding under various conditions. An important use of the assay could be to assess the ability of various small molecules to interrupt this interaction, which could potentially be
developed as novel therapeutics for the treatment of diseases that overexpress CXCR4. In addition, the assay could be applied to a variety of experiments in order to further elucidate the mechanisms by which the β-arrestin-1/STAM-1 complex interacts with of other proteins to modulate the sorting of CXCR4 on endosomes.
The assay designed in this project utilizes bioluminescence resonance energy transfer (BRET) as a measurement of the interaction status by the co-expression of STAM1-Rluc (Renilla luciferase) with β-arrestin1-YFP. The addition of a coelenterazine(h) substrate induces the emission of light from Rluc, which is absorbed by the yellow fluorescent protein (YFP) and emitted at a different wavelength, if the proteins are interacting. It was expected that expression of the two fusion proteins would yield a relatively low BRET signal in cells that had not been stimulated with CXCL12. However, following CXCL12 stimulation, a significantly higher BRET signal was expected, since CXCR4 is rapidly internalized to endosomes upon ligand binding.
After attempting to use BRET to detect an interaction between two proteins whose interaction has already been shown by BRET, and using BRET to examine the interaction between two proteins that are not expected to interact, it was determined that BRET measurements between STAM1-Rluc and β-arrestin1-YFP were likely showing a slight interaction between the two proteins. No differences were seen in BRET between cells stimulated with CXCL12 or vehicle. It is concluded from the obtained results that further optimization steps are required for the assay described here to be amenable to any future studies of the interaction between STAM-1 and β-arrestin-1.
Buhrmaster, James, "Creating a BRET Assay to Monitor the Interaction between β-Arrestin-1 and STAM-1" (2017). Master's Theses. 3663.
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Copyright © 2017 James Buhrmaster