Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology and Experimental Therapeutics

Abstract

G protein-coupled receptor (GPCR) sorting into the degradative pathway is important for attenuating signaling. Perturbations in this process can manifest in a variety of diseases. Upon agonist activation of the chemokine receptor CXCR4, a GPCR, it is rapidly ubiquitinated, internalized to endosomes and sorted for degradation in lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. This process culminates in attenuation of CXCR4 signaling. CXCR4 overexpression and increased CXCR4 signaling have been associated with several pathologies including immune deficiency disorders and over 23 cancers. Yet the mechanisms governing the regulation of CXCR4 signaling remain elusive.

CXCR4 is ubiquitinated by the HECT-domain ligase AIP4 at the plasma membrane. AIP4 is localized on early endosomes and regulates CXCR4 sorting by modulating the activity of the ESCRT machinery. In particular, ESCRT-0 ubiquitination has been shown to be linked to the efficiency by which CXCR4 is sorted for lysosomal degradation. However, mechanistic insight is lacking and the precise role of AIP4 in these processes remains poorly defined.

The objective of this project is to gain a greater understanding of the mechanisms mediating AIP4 regulation of CXCR4 degradation. AIP4 is known to interact with other E3 ligases, including DTX1 and Cbl-c, but whether these E3 ligases or others are involved in CXCR4 sorting is not known. Here, we show for the first time that the RING-domain ubiquitin ligase Deltex-3-like (DTX3L) mediates CXCR4 sorting from early endosomes to lysosomes. Using several biochemical and immunochemical techniques, we show that upon CXCR4 activation DTX3L localizes to early endosomes where it directly interacts with and inhibits the activity of the AIP4. Thereby, limiting the extent to which ESCRT-0 is ubiquitinated while promoting CXCR4 sorting for lysosomal degradation. Therefore, we have defined a novel role for DTX3L in GPCR endosomal sorting and propose that DTX3L may play a broad role in endosomal sorting. In addition, our data reveal an unprecedented link between two distinct ubiquitin ligases to control the activity of the ESCRT machinery. Overall, these findings may prove beneficial in developing strategies to modulate CXCR4 levels and be broadly applicable to CXCR4-related pathologies.

Creative Commons License

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

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