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G-protein-coupled receptors (GPCRs) constitute the largest but the most divergent class of cell surface proteins. Although they are thought to share a common 3D-structure composed of seven transmembrane helical domains, they can be activated by extracellular signals as diverse as light, peptides, proteins, lipids, organic odorants, taste molecules, nucleotides or nucleosides. They are involved in an extraordinarily large number of physiological functions and are therefore potential drug targets for many human diseases. During the last decade various GPCRs have been successfully expressed in S. cerevisiae. Yeast is an attractive expression system because it offers the genetic engineering tools typical of a microorganism while possessing an eukaryotic type of secretory pathway and post-translational machinery. This host is particularly attractive for in-vivo manipulation of these receptors due to the high homology between the yeast pheromone signaling pathway and that of mammalian GPCRs. When expressed in yeast, mammalian GPCRs have been shown to couple functionally to either the endogenous yeast Galpha (Gpa1), or co-expressed mammalian Galpha subunits (wild-type or chimeric), and are characterized by a similar pharmacology in response to agonists or antagonists as in native cells. Heterologous expression of wild type or mutant GPCRs in S. cerevisiae allows a rapid assessment of their ability to detect and transduce extracellular stimulations, through the use of a reporter system. Furthermore, this approach is amenable to high-throughput screening of new drugs, which would provide a determinant advantage in the field of therapeutic research, and also for investigation of the still unknown ligands of orphan receptors. This review will focus on the latest developments of yeast-based technology to screen for potential GPCR agonists/antagonists.

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the ␣ subunit of the G protein G i1 and the porcine ␣ 2A -adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous G i ␣. Half-maximal stimulation of the GTPase activity of endogenous G i was achieved with lower concentrations of agonist. Although the K m for GTP of the fusion protein-linked G i was lower than for the endogenous G protein, V max measurements demonstrated that adrenaline activated some 5 mol of endogenous G i /mol of fusion proteinlinked G i . The isolated ␣ 2A -adrenoreceptor could activate G s ; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous G i ␣ was greater than for the fusion protein-constrained C351G G i1 ␣. ␣ 2A -Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated ␣ 2A -adrenoreceptor-C351G G i1 ␣ fusion proteinexpressing cells, but these signals were abolished following pertussis toxin treatment.These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry. G protein-coupled receptors (GPCRs)1 initiate vectorial signal transduction cascades via activation of heterotrimeric G proteins and the subsequent regulation of effector enzymes (1, 2). These function to amplify cellular response to the presence of low concentrations of extracellular mediators. As part of this process, agonist-occupied GPCRs have the capacity to catalytically activate G proteins. Some 20 distinct G protein ␣-subunits are known, and in many cases they are highly similar in sequence. Many cells co-express a considerable number of distinct but highly related G proteins. As a novel means to examine the functional interactions of a GPCR with a single G protein, we recently generated a fusion protein between the ␣ 2A -adrenoreceptor and the ␣-subunit of G i1 (3, 4). Since G i1 is a member of the subfamily of G proteins that can be modified by ADP-ribosylation catalyzed by pertussis toxin (5) and a number of these G proteins are routinely co-expressed by all cells (5), we used a modified version of G i1 ␣ (C351G G i1 ␣) that is resistant to the action of pertussis toxin (6) to generate the fusion protein. Following transient expression of the fusion protein in COS-7 cells, we were able to ...

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