| Title: | Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast alpha-Factor Receptor |
| Address: | "Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA. Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY 11794-5222, USA" |
| ISSN/ISBN: | 2218-273X (Electronic) 2218-273X (Linking) |
| Abstract: | "The Saccharomyces cerevisiae alpha-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures" |
| Keywords: | "Cysteine/metabolism GTP-Binding Proteins/metabolism Receptors, G-Protein-Coupled/genetics/metabolism *Receptors, Mating Factor/chemistry/genetics/metabolism *Saccharomyces cerevisiae/metabolism *Saccharomyces cerevisiae Proteins/metabolism constitutively;" |
| Notes: | "MedlineDumont, Mark E Konopka, James B eng R01 AI047837/AI/NIAID NIH HHS/ R01 GM116048/GM/NIGMS NIH HHS/ R01GM084083, RO1GM059357, R01GM114974, U54GM074899, and U54GM094611 (to M.E.D.) and R01AI047837 and RO1GM116048 (to J.B.K)./NH/NIH HHS/ Research Support, N.I.H., Extramural Review Switzerland 2022/06/25 Biomolecules. 2022 May 30; 12(6):761. doi: 10.3390/biom12060761" |
