Functional Analysis of Heterologous GPCR Signaling Pathways in Yeast Graham Ladds, Alan Goddard and John Davey Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK TRENDS in Biotechnology Vol.23 No.7 July 2005 Benesh Joseph and Watanabe Shinpei Review article introducing the possibilities of using yeast to analyze GPCRs, focusing on the budding yeast Saccharomyces cerevisiae and recent developments using the fission yeast Schizosaccharomyces pombe.
Diversity of GPCRs, G proteins and their Signaling Pathways
Pharmacological Importance of GPCRs ・ ~30% of marketed drug act on GPCRs ・ sales: US$23 billion/Year
Signaling Pathway GPCR modifications Yeast has two GPCR signaling pathway – one that mediates pheromone response and a second one that senses glucose. Although either could be exploited there are no reports on coupling of non-yeast GPCRs to the glucose sensing pathway,
Modifying G α Protein for expression in Yeast The yeast G α (Gpa1) interacts with several non-yeast GPCRs but the interaction can be inefficient. Chimeric G α subunits were therefore developed to incorporate receptor binding properties of mammalian subunits into a Gpa1 subunit that retains efficient interaction with the yeast G. Gpa 1 G α Gpa 1/G α Gpa 1/G α can be coupled to various GPCRs by attaching the C-terminal region of the corresponding G α to Gpa1 Inefficient interaction with heterologous GPCR Efficient interaction with heterologous GPCR
Most GPCRs can be expressed unmodified but some get retained or degraded within
The stability or activity of some receptors can also be increased by
By adding a cleavable leader sequence for transport to the plasma membrane.
2. Removing regions not required for interacting with the ligand or G protein.
Wess J. Pharmacol. Ther. Vol. 80, No. 3, pp. 231–264, 1998 Deleting amino acids except the few near the membrane in the i4 loop gave Enhanced expression of the receptor Mascarinic Receptor
Some receptors require co-expression of R eceptor A ctivity M odifying P roteins ( RAMPs ) to ensure activity or to create distinct pharmacological profiles. Because these interacting proteins are likely to be absent in yeast, their importance for the functionality of the receptor can be studied individually in yeast C,D,E – C alcitonin R eceptor L ike R eceptor ( CRLR or CGRP Receptor ): R1, R2, R3 – R eceptor A ctivity M odifying P roteins ( RAMP): CGRP – C alcitonin G ene R elated P rotein Sexton M P et.al Cellular Signaling 13 (2001) 73± 83
1 2 3 Ligand Library New Drugs Ligand Screening for New Drug
Defining Coupling Specificity between GPCR and G α The genetic amenability and high efficiency of transformation of the yeast means there is almost no limit to the number of individuals that can be screened to select the one with desired characteristics from a large library of mutants. Methionine 145 in the i2 loop determine whether V2 Vasopressin receptor binds to Gs or Gq. Deletion of any of the residues highlighted in light blue (Ile141–Pro144) completely abolished V2 receptor function probably by interfering with proper receptor folding. On the other hand, deletion of any of the residues highlighted in yellow (Leu146–Arg149) had no effect on the G protein coupling properties of the V2 receptor. V2 Vasopressin receptor i2 loop Authors reached the conclusion on analyzing 30,000 mutant receptor libraries for their ability to bind with Gpa1 p/G Q (the receptor normally binds to G α s or Gpa1 p/ G α s )!! Erlenbach,I et al.JBC. Vol. 276, No. 31, Issue of August 3, pp. 29382–29392, 2001
Autocrine Selection in Yeast: Co-expression of Receptor and Ligand NATURE BIOTECHNOLOGY VOLUME 16 DECEMBER 1998 formyl peptide receptor like-1 (FPRL-1) Receptor was an ORPHAN GPCR and the functional significance was unknown Now is proven that FPRL-1 binds with many peptide ligands like V3 region of the HIV-1 envelope gp120, human prion peptide etc.. and mediate Chemo taxis and inflammatory response Elagoz A et al. British Journal of Pharmacology (2004) 141, 37–46
Nature Reviews Drug Discovery 3, 577- 626 (2004) Characterization of the natural ligand of an Orphan GPCR Yeast Cell
Regulators of GPCRs – Receptor Kinases GRK – G protein R eceptor K inase: PDE - Phosphodiesterase Kohout A T et. al. Mol. Pharmacol. 63:9–18, 2003 GRK expression in Yeast causes Reduced signaling from Corresponding GPCR.
R egulators of G protein S ignalling ( RGSs ) RGS bind to activated G α Subunits to accelerate GTP hydrolysis and Inactivation . In mammalian Cells there are multiple receptors, G proteins and RGSs. Analysis of interaction between individual GPCR- G α –RGS combinations is often simpler in yeast in which components can be Easily manipulated. Nature Reviews Drug Discovery 1; 187-197 (2002);
A ctivators of G protein S ignaling ( AGS ) and their Regulators AGS3 GDP GTP AGS1 (act as GEF) AGS2 – destabilize G protein complex AGS3 AGS4 + The potential effects of AGS proteins on G protein signaling are obvious but it remains unclear what might regulate their activity in mammalian cells. It is highly likely that yeast will continue to have a role in understanding this important group of regulators GEF – Guanine Nucleotide Exchange Factor +
Discovering Peptide Inhibitors of MAP Kinase Pathway MAP Kinase Cascade Identifying peptide inhibitors of yeast enzyme 1. to use yeast two-hybrid analysis to select peptides that bind to the target enzyme. select peptides that bind to the target enzyme and then use a functional assay to identify which of these inhibit signaling to express a randomized library of peptide and to identify those able to inhibit signaling through the pheromone pathway 2. Yeast has a similar MAP Kinase pathway as that of higher eukaryotes
Other Yeast Systems – Schizosaccharomyces pombe Schizosaccharomyces pombe isolated from randomly mutagenized population of GPCR receptors and assayed for colony formation on ura - medium supplemented with different amounts of pheromone .
Ligand-directed trafficking in G α transplanted Schizosaccharomyces pombe Ligand-directed trafficking in G α -transplant yeast strains. Receptors for corticotropin-releasing factor (CRF) were expressed in a series of Sz. pombe LacZ reporter strains, each containing a different G α -transplant. Cells were exposed to CRF-related ligands urocortin (UCN), UCN II and UCN III at 10 -6 M before being assayed for b-galactosidase activity. Different combinations of ligand and receptor activated the various G α -transplants to different extents. The G α -transplants are created by replacing the extreme C-terminus of the yeast Gpa1 with the correspond residues of the human G α subunits indicated. CRF-R1a, corticotropin-releasing factor receptor type 1; CRF-R2b, corticotropin-releasing factor receptor type 2b. A GPCR could have different pharmacological profiles depending on which G protein is activated, and that the same GPCR could have different roles in different cell types depending on the local G protein environment.
The experimental tractability of the yeast, particularly the ability to screen large numbers of randomly generated mutants for isolates with particular phenotypes, enable the use of approaches that cannot be used in mammalian systems. Performing the work in a host in which all endogenous GPCRs and G α subunits can be eliminated provides clear and unambiguous results. The receptors, their G proteins, the intracellular signaling components and their regulators and modulators are all accessible for analysis. Conclusions