The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation

Uddin, Mohammad Moin; Hauser, Melinda; Naider, Fred; Becker, Jeffrey M.

doi:10.1016/j.bbamem.2015.12.017

Cited by 16 publications

(5 citation statements)

References 73 publications

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“…This strategy has been used previously in our lab to determine the involvement of TM regions in dimerization [60]. In a recent report we showed that the N-terminus plays an important role in the regulation of Ste2p signaling [69]. The finding herein begins to reveal at the residue level a possible basis for this regulatory control.…”

Section: Discussionmentioning

confidence: 70%

Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p

Uddin

Naider

Becker

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The Saccharomyces cerevisiae α-factor receptor Ste2p has been used extensively as a model to understand the molecular mechanism of signal transduction by G protein-coupled receptors (GPCRs). Single and double cysteine mutants of Ste2p were created and served as surrogates to detect intramolecular interactions and dimerization of Ste2p using disulfide cross-linking methodology. When a mutation was introduced into the phylogenetically conserved tyrosine residue at position 26 (Y26C) in the N-terminus of Ste2p, dimerization was increased greatly. The amount of dimer formed by this Y26C mutant was greatly reduced by ligand binding even though the ligand binding site is far removed from the N-terminus; the lowering of the dimer formation was consistent with a conformational change in the N-terminus of the receptor upon activation. Dimerization was decreased by double mutations Y26C/V109C or Y26C/T114C indicating that Y26 is in close proximity to V109 and T114 of extracellular loop 1 in native Ste2p. Combined with earlier studies, these results indicate previously unrecognized roles for the N-terminus of Ste2p, and perhaps of GPCRs in general, and reveal a specific N-terminus residue or region, that is involved in GPCR signaling, intrareceptor interactions, and receptor dimerization.

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Section: Discussionmentioning

confidence: 70%

Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p

Uddin

Naider

Becker

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The deletion of residues two through 10 of Ste2p resulted in an overexpression of the receptor on the cell surface, thus implying that this region, or its influence on the conformation of the receptor, acted to negatively regulate receptor expression [ 152 ]. In addition, we had shown previously that residues 20 through 30 formed a β-sheet [ 153 ], and that residues in extracellular loop one (EL1) formed a 3 10 helix [ 154 ].…”

Section: Genetic Manipulation Of the Receptor Structurementioning

confidence: 99%

A Paradigm for Peptide Hormone-GPCR Analyses

Naider

Becker

2020

Molecules

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Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

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“…The extracellular domains, intracellular domains, and cytoplasmic C-terminal regions correspond to those predicted for the S. cerevisiae Ste2 and Ste3 (45), as well as P. carinii Map3 receptors (18).…”

Section: Methodsmentioning

confidence: 55%

Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors

Luraschi

Richard

Almeida

et al. 2019

mBio

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The genus Pneumocystis encompasses fungal species that colonize mammals’ lungs with host specificity. Should the host immune system weaken, the fungal species can cause severe pneumonia. The life cycle of these pathogens is poorly known, mainly because an in vitro culture method has not been established. Both asexual and sexual cycles would occur. Trophic cells, the predominant forms during infection, could multiply asexually but also enter into a sexual cycle. Comparative genomics revealed a single mating type locus, including plus and minus genes, suggesting that primary homothallism involving self-fertility of each strain is the mode of reproduction of Pneumocystis species. We identified and analyzed the expression of the mam2 and map3 genes encoding the receptors for plus and minus pheromones using reverse transcriptase PCR, in both infected mice and bronchoalveolar lavage fluid samples from patients with Pneumocystis pneumonia. Both receptors were most often concomitantly expressed during infection, revealing that both pheromone-receptor systems are involved in the sexual cycle. The map3 transcripts were subject to alternative splicing. Using immunostaining, we investigated the presence of the pheromone receptors at the surfaces of Pneumocystis cells from a patient. The staining tools were first assessed in Saccharomyces cerevisiae displaying the Pneumocystis receptors at their cellular surface. Both receptors were present at the surfaces of the vast majority of the cells that were likely trophic forms. The receptors might have a role in mate recognition and/or postfertilization events. Their presence at the cell surface might facilitate outbreeding versus inbreeding of self-fertile strains. IMPORTANCE The fungi belonging to the genus Pneumocystis may cause severe pneumonia in immunocompromised humans, a disease that can be fatal if not treated. This disease is nowadays one of the most frequent invasive fungal infections worldwide. Whole-genome sequencing revealed that the sexuality of these fungi involves a single partner that can self-fertilize. Here, we report that two receptors recognizing specifically excreted pheromones are involved in this self-fertility within infected human lungs. Using fluorescent antibodies binding specifically to these receptors, we observed that most often, the fungal cells display both receptors at their surface. These pheromone-receptor systems might play a role in mate recognition and/or postfertilization events. They constitute an integral part of the Pneumocystis obligate sexuality within human lungs, a cycle that is necessary for the dissemination of the fungus to new individuals.

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The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation

Cited by 16 publications

References 73 publications

Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p

Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p

A Paradigm for Peptide Hormone-GPCR Analyses

Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors

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