Biased Constitutive Activity in the Uveal Melanoma Oncogene CYSLTR2 is Unique in CYSLTR2 Germline and Pan-Cancer Human Variome

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor (GPCR) cysteinylleukotriene receptor 2. The mutant CYSLTR2 encodes for CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. In this work, we characterized the functional properties of CysLTR2-L129Q. CysLTR2 signals through the Gq/11/PLC-β pathways, so using a homogenous time resolved fluorescence (HTRF) IP1 accumulation assay, we show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits β-arrestin as shown by a bioluminescence resonance energy transfer 2 (BRET2) based β-arrestin recruitment assay. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping β-arrestin-mediated downregulation. CYSLTR2 is the first known example of a GPCR driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma. Furthermore, we learned that CysLTR2 is a significantly mutated GPCR in several other cancers as well. We identified \u3e100 CYSLTR2 missense variants of unknown significance (VUS) in human cancer genomes from available cancer databases, as well as another \u3e100 CYSLTR2 single-nucleotide polymorphisms (SNPs) from exome sequence data. Here, we introduce a proof-of-concept, experimental, activity-based profiling pipeline to systematically assess the mutational landscape of CYSLTR2. We use a single transfection mixture of receptor-encoding DNA and HEK293T cells is used to characterize all variants for expression level, basal and agonist-stimulated G protein signaling, and basal and agonist-stimulated β-arrestin recruitment. The CysLTR2-L129Q mutation causing uveal melanoma has a unique phenotype among all cancer-associated variants. It is highly constitutively active with gain-of-function (GoF) in basal Gq/11-PLC-β signaling and loss-of-function (LoF) in agonist-dependent signaling, while only poorly recruiting β-arrestin. Furthermore, we found that about 21% of the variants show no detectable activity and are basically indistinguishable from mock-transfected controls, suggesting that a large portion of these mutations are damaging. A further 21% lose 50% of activity as normalized to WT (100%), and another ten percent are nonsense and frameshift variants. This means that about 50% of total somatic mutations of CYSLTR2 have a LoF phenotype, which points to a tumor suppressor function following the famous “20/20” rule

High-affinity binding of chemokine analogs that display ligand bias at the HIV-1 coreceptor CCR5

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound