Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes

Abstract

G protein-coupled receptors (GPCRs) comprise the largest class of membrane proteins in the human genome, with a common denominator of seven-transmembrane domains largely conserved among eukaryotes. Yeast is naturally armoured with three different GPCRs for pheromone and sugar sensing, with the pheromone pathway being extensively hijacked for characterising heterologous GPCR signalling in a model eukaryote. This review focusses on functional GPCR studies performed in yeast and on the elucidated hotspots for engineering, and discusses both endogenous and heterologous GPCR signalling. Key emphasis will be devoted to studies describing important engineering parameters to consider for successful coupling of GPCRs to the yeast mating pathway. We also review the various means of applying yeast for studying GPCRs, including the use of yeast armed with heterologous GPCRs as a platform for (i) deorphanisation of orphan receptors, (ii) metabolic engineering of yeast for production of bioactive products and (iii) medical applications related to pathogen detection and drug discovery. Finally, this review summarises the current challenges related to expression of functional membrane-bound GPCRs in yeast and discusses the opportunities to continue capitalising on yeast as a model chassis for functional GPCR signalling studies.

Serotonin GPCR-based biosensing modalities in yeast

“…Likewise, knockout of SST2, a negative regulator of GPA1, and FAR1, an inducer of cell cycle arrest during mating, have been key steps to increase heterologous GPCR signalling in yeast, as demonstrated previously (Dohlman et al, 1996;Price et al, 1995), ultimately enabling the development of whole-cell biosensors based on >50 GPCRs (Kapolka et al, 2020;Lengger and Jensen, 2020;Shaw et al, 2019).…”

“…While these modules constitute the core GPCR signalling, a dearth of knowledge challenges our understanding of how the approx. 800 GPCRs encoded in the human genome couple through 16 different Gα subunits (Pándy-Szekeres et al, 2018), notwithstanding the structure-affinity relationship between the great diversity of ligands and the GPCRs which have evolved to respond to them, including light, hormones, and small molecules, like serotonin ( (Lengger and Jensen, 2020;UniProt Consortium, 2021)).…”

“…For more than three decades yeast has served as a platform for studying human GPCRs (Brown et al, 2000;King et al, 1990) with great potential in both medical and biotechnological application areas (Lengger and Jensen, 2020). The vast majority of GPCR studies in yeast are based on the mating pathway naturally activated by pheromone through the yeast mating-factor GPCRs Ste2/3 (Versele et al, 2001).…”

“…The vast majority of GPCR studies in yeast are based on the mating pathway naturally activated by pheromone through the yeast mating-factor GPCRs Ste2/3 (Versele et al, 2001). Upon ligand activation, successful coupling of a heterologous GPCR with the yeast mating pathway can occur through coupling to the yeast Gα protein GPA1, which subsequently activates the mitogenactivated protein (MAP) kinase cascade consisting of Ste5/Ste7/Ste11, ultimately resulting in the activation of 100s of pheromone responsive genes (Lengger and Jensen, 2020). While a few studies successfully proved coupling of human GPCRs to GPA1 (Ehrenworth et al, 2017;King et al, 1990;Mukherjee et al, 2015), a key contribution to potentiate coupling of heterologous GPCRs to the yeast mating pathway was the discovery of improved coupling when exchanging the C-terminal five amino acids in the G part of GPA1 with complementary 5-mer human G signatures, also referred to as chimeric Gα proteins (Brown et al, 2000;Conklin and Bourne, 1993;Nakamura et al, 2015;Shaw et al, 2019).…”

A computational model for GPCR-ligand interaction prediction

“…GPCRs are classified in five distinct groups (i.e., glutamate [G], rhodopsin-like [R], adhesion [A], frizzled/taste [F] and secretin [S]) [ 6 ]. Moreover, GPCRs are the biggest receptor family among all the cell surface receptors [ 7 ]. A significant part of GPCR-targeted drug development is related to an interaction between GPCRs and other molecules (e.g., natural ligands, synthetic agonists, and other proteins) [ 4 ].…”