A Humanized CB1R Yeast Biosensor Enables Facile Screening of Cannabinoid Compounds.

Yeast expression of human G-protein-coupled receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. Cannabinoid receptor type 1 (CB1R) is of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane modulates its performance. Using an engineered cannabinoid biosensor, we demonstrate that large libraries of synthetic cannabinoids and terpenes can be quickly screened to elucidate known and novel structure-activity relationships. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing therapeutic molecules.

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols.

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.