Abstract
In cancer, G protein-coupled receptors (GPCRs) are involved in tumor progression and metastasis. In this study we particularly examined one GPCR, the adenosine A2B receptor. This receptor is activated by high concentrations of its endogenous ligand adenosine, which suppresses the immune response to fight tumor progression. A series of adenosine A2B receptor mutations were retrieved from the Cancer Genome Atlas harboring data from patient samples with different cancer types. The main goal of this work was to investigate the pharmacology of these mutant receptors using a ‘single-GPCR-one-G protein’ yeast assay technology. Concentration-growth curves were obtained with the full agonist NECA for the wild-type receptor and 15 mutants. Compared to wild-type receptor, the constitutive activity levels in mutant receptors F141L4.61, Y202C5.58 and L310P8.63 were high, while the potency and efficacy of NECA and BAY 60–6583 on Y202C5.58 was lower. A 33- and 26-fold higher constitutive activity on F141L4.61 and L310P8.63 was reduced to wild-type levels in response to the inverse agonist ZM241385. These constitutively active mutants may thus be tumor promoting. Mutant receptors F259S6.60 and Y113F34.53 showed a more than one log-unit decrease in potency. A complete loss of activation was observed in mutant receptors C29R1.54, W130C4.50 and P249L6.50. All mutations were characterized at the structural level, generating hypotheses of their roles on modulating the receptor conformational equilibrium. Taken together, this study is the first to investigate the nature of adenosine A2B receptor cancer mutations and may thus provide insights in mutant receptor function in cancer.
Highlights
G protein-coupled receptors (GPCRs) are a family of membranebound proteins that have seven-transmembrane (7TM) domains, connected by three intracellular (IL) and three extracellular (EL) loops, an extracellular amino terminus, and an intracellular carboxyl terminus (Vassilatis et al, 2003)
A2B receptor identified from The Cancer Genome Atlas (TCGA) (Broad Institute TCGA Genome Data Analysis Center, 2016) were examined in the S.cerevisiae system to improve our understanding of the mechanism of receptor activation in relation to cancer progression
15 cancer-related somatic mutations on the adenosine A2B receptor were retrieved from TCGA and characterized in a robust yeast system
Summary
G protein-coupled receptors (GPCRs) are a family of membranebound proteins that have seven-transmembrane (7TM) domains, connected by three intracellular (IL) and three extracellular (EL) loops, an extracellular amino terminus, and an intracellular carboxyl terminus (Vassilatis et al, 2003). In total approximately 800 GPCRs are present in the human genome which can be subdivided in five main families, namely glutamate, rhodopsin, adhesion, frizzled/taste, and secretin, according to the GRAFS classification system (Fredriksson et al, 2003). GPCRs have been relatively underappreciated in preclinical oncology, where primary focus over the last two decades has been on kinases due to their central role in the cell cycle. There is a growing body of evidence showing a more prominent role of GPCRs in all phases of cancer (Lappano and Maggiolini, 2012). Recent work has shown that the function of GPCRs present in patient isolates is altered due to mutation, and GPCRs are mutated in an estimated 20% of all cancers (Kan et al, 2010; Watson et al, 2013)
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