Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Thomas P. Sakmar
Keywords
G protein-coupled receptor, membrane receptor, pharmacology, proteolysis-targeting chimeras, uveal melanoma, CysLTR2
Abstract
G protein-coupled receptors (GPCRs) are a family of heptahelical transmembrane proteins that can modulate various cellular signaling pathways and therefore are attractive drug targets. Despite high mutational rates of GPCRs in cancer there are few oncology FDA-approved anti-GPCR drugs, leaving a gap in the therapeutic landscape. Drug discovery platforms such as targeted protein degradation (TPD), provide opportunities to target oncogenic proteins by direct modulation of protein expression. TPD methodologies can be used to target oncogenic GPCRs that are overexpressed or constitutively active, as in the case of constitutively active mutants (CAMs), which have roles in cancer progression. TPD methods include proteolysis targeting chimeras (PROTACs), which are heterobifunctional molecules where one end has a ligand for the protein-of-interest (POI), and the other end has an E3 ligase recruiting ligand. PROTACs take advantage of the ubiquitin-proteasome system (UPS) by forcing a POI in proximity to an E3 ligase through the formation of a ternary complex between the POI, PROTAC, and E3 ligase. Then promoting the transfer of ubiquitin to the POI as a signal for proteasomal degradation. PROTACs have been applied to multiple soluble proteins, however, recently this drug discovery platform is being applied to membrane proteins such as GPCRs. Studies using conditional degron tags (CDTs) and specific PROTACs provide an initial starting point showing the feasibility of PROTACs as a possible drug discovery platform for GPCRs. In this work, we further validate the use of PROTAC technology, specifically the HaloPROTAC3 and HaloTag7 CDT system, to degrade GPCRs as a proof-of-concept study. We created a panel of GPCRs with a C-terminal HaloTag7 to promote the intracellular binding orientation of the HaloPROTAC3 and, therefore, promote E3 ligase recruitment, ubiquitination, and degradation. We used both a split-luminescence complementation assay and immunoblot assay to see the effects of the HaloPROTAC3 on the protein abundance. Additionally, we used a secondary messenger d-myo-inositol-1-phosphate (IP1) accumulation assay to measure the changes in activation of the Gaq signaling pathway with the addition of the HaloPROTAC3. We observed through these assays that the protein abundance and the signaling of each GPCR decreased with the addition of the HaloPROTAC3. Addition of an inactive enantiomer HaloPROTAC3 showed a rescue in protein abundance and signaling, indicating that the decrease in protein abundance and signaling is due to the recruitment of the von Hippel-Lindau (VHL) E3 ligase. The recruitment of the E3 ligase suggests that the receptors are ubiquitinated and degraded causing the decrease in protein abundance and signaling. This work shows that PROTAC technology can be more widely applied to GPCRs. Additionally, we used this platform to test the degradation of a disease relevant GPCR, cysteinyl leukotriene receptor 2 (CysLTR2), which is associated with uveal melanoma (UM). UM is an eye cancer that develops in melanocytes with high mortality rates due to high incidence of metastasis. In a subset of cases, the oncogenic driver is the activating mutation in the gene CYSLTR2, which encodes a substitution of a Leu to Gln at position 129 in the GPCR CysLTR2. CysLTR2-L129Q has a gain-of-function phenotype because it signals in the absence of an agonist ligand and can escape downregulation. Therefore, development of a PROTAC could provide therapeutic efficacy through the degradation of the constitutively active receptor. Using a similar HaloPROTAC3 proof-of-concept study, we showed that the protein abundance and constitutive activity can be decreased with the addition of the HaloPROTAC3. Additionally, the inactive enantiomer HaloPROTAC3 showed a rescue in protein abundance and constitutive activity. Therefore, the decrease from the HaloPROTAC3 is due to the recruitment of the VHL E3 ligase. We then confirmed the formation of a ternary complex between CysLTR2-L129Q, the HaloPROTAC3, and the VHL E3 ligase. Furthermore, we used a neddylation inhibitor, a proteasomal inhibitor, and a lysosomotropic agent to confirm the mechanism of ubiquitination and proteasomal degradation. Lastly, using primary human melanocytes we showed that the HaloPROTAC3 results in a decrease in protein expression of CysLTR2-L129Q in pathologically relevant cells. Overall, this work shows the feasibility of using PROTAC technology to degrade GPCRs and specifically an oncogenic CAM GPCR, illuminating the possibility of therapeutic anti-GPCR PROTACs.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Saca, Victoria, "Induced Degradation of G-Protein-Coupled Receptors Through Proteolysis Targeting Chimeras" (2025). Student Theses and Dissertations. 840.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/840
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy