Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling

Emilie Ceraudo,Mizuho Horioka,Jordan M Mattheisen,Tyler D Hitchman,Amanda R Moore,Manija A Kazmi,Ping Chi,Yu Chen,Thomas P Sakmar,Thomas Huber

doi:10.1074/jbc.ra120.015352

Abstract

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 cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the 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. Here, we characterize the functional properties of CysLTR2–L129Q. 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. 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 G protein–coupled receptor 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.

Highlights

The superfamily of G protein–coupled receptors (GPCRs) is the largest gene family encoding cell-signaling transmembrane proteins, and approximately one-quarter of 400 nonolfactory GPCRs are therapeutic drug targets
We show that cysteinyl-leukotriene receptor 2 (CysLTR2)–L129Q is a constitutively active mutant (CAM) receptor that strongly couples to Gq/11 cellular signaling pathways
Phospholipase C-β (PLCβ) is the classical effector of Gq/11 and results in receptor-stimulated phosphoinositide hydrolysis that is conveniently monitored as an accumulation of D-myo-inositol-1-phosphate (IP1) in the presence of lithium chloride (LiCl) [10]

Summary

Results and discussion

To characterize the functional phenotype of CysLTR2– L129Q, we first determined the agonist-dependent signaling for CysLTR2–L129Q and CysLTR2 WT. After 100 min, the basal IP1 accumulation of CysLTR2–L129Q kept increasing, whereas the ligand-dependent signaling of the WT receptor reached a plateau (Fig. S1B). We generated fusion constructs of CysLTR2 WT and CysLTR2–L129Q, with a version of GFP (GFP10) at the Cterminus These fusion constructs enable quantification of basal and agonist-dependent Gq/11 cellular signaling and βarrestin recruitment activity under comparable conditions. CysLTR2–L129Q (maroon) shows higher basal β-arrestin recruitment, corresponding to 13.5% constitutive activity, and no response to agonist. The shapes of the time courses were similar when comparing samples expressing β-arrestin1–RLuc and β-arrestin2– RLuc, but the peak increase seen for β-arrestin2–RLuc was almost twice that of β-arrestin1–RLuc (Fig. S2) Such a biphasic BRET2 β-arrestin recruitment time course is typical for GPCRs with “class A” β-arrestin–recruitment phenotype

A Sigmoidal dose-response function:

CysLTR2-V2 L129Q

Experimental procedures