Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins
UNSPECIFIED. (2003) Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins. MOLECULAR MICROBIOLOGY, 47 (3). pp. 781-792. ISSN 0950-382XFull text not available from this repository.
G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R-1A receptor than against the CRF-R-2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R-2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R-1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry
Q Science > QR Microbiology
|Journal or Publication Title:||MOLECULAR MICROBIOLOGY|
|Publisher:||BLACKWELL PUBLISHING LTD|
|Number of Pages:||12|
|Page Range:||pp. 781-792|
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