Download or read book Interaction of a G Protein coupled Receptor Ste2p of Saccharomyces Cerevisiae with Its Ligand and Its G protein Alpha Subunit written by Li-Yin Huang and published by . This book was released on 2011 with total page 220 pages. Available in PDF, EPUB and Kindle. Book excerpt: The G protein-coupled receptor (GPCR) family is composed of hundreds of members and is expressed in eukaryotes. Each GPCR has seven transmembrane domains and is in charge of sensing changes from the environment, transducing signals, and activating a series of biological responses. The signal transduction pathway of the receptor starts from sensing outside signal and then activates G proteins. This signaling requires a tight control for activation without which impaired cellular function leads to pathology. We have used the pheromone alpha-factor receptor (Ste2p) of the yeast Saccharomyces cerevisiae as a model system to understand ligand binding, receptor activation, and G protein interaction. One method we have used to study ligand binding is to incorporate the photo-reactive crosslinker p-benzoyl-L-phenylalanine (Bpa) into Ste2p to capture alpha-factor. This powerful tool requires the incorporation of Bpa, an unnatural amino acid, into Ste2p by a special genetic manipulation designed in the lab of Peter Schulz (Scripps Institute) and adapted by our lab for Ste2p. Another method to study ligand binding that we have adapted for use in our system is to incorporate a chemical crosslinker [3,4-dihydroxylphenylacetyl (DHPA)] into alpha-factor for periodatemediated crosslinking to Ste2p. The interacting domain between alpha-factor and transmembrane domain 2 to 3 of Ste2p was identified after DOPAC crosslinking, cyanogen bromide digestion and MALDI-TOF mass spectrometry. After ligand binding, signal transduction is mediated by the interaction of activated Ste2p with its G protein (Gpa1p). We studied this interaction by replacing natural residues in the intracellular loop 3 of Ste2p and C-terminal end of Gpa1p with cysteine and then determining disulfide crosslinking between Ste2p and Gpa1p. Some residues were found to be in close proximity and displayed different interacting patterns due to conformational changes of the receptor upon ligand binding. The information we gathered here allows us to understand more about the physical interactions of alpha-factor, Ste2p, and Gpa1p and provides us insights about the initiation and activation of the signal transduction pathway of a peptide ligand receptor.

Download or read book Saccharomyces Cerevisiae G Protein Coupled Receptor Ste2p Interactions with Its Ligand alpha factor and Cognate G alpha Protein Gpa1p written by George Kwabena Essien Umanah and published by . This book was released on 2009 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Saccharomyces cerevisiae alpha-factor receptor, Ste2p, belongs to the G protein-coupled receptors (GPCRs), a class of integral membrane proteins that are characterized by seven-transmembrane (TM) domains. Ste2p-alpha-factor pair has been used extensively as a paradigm for investigating GPCRs structure and function. Upon binding of alpha-factor to Ste2p, a signal is transduced via an associated guanine-nucleotide binding protein, Gpa1p, initiating a cascade of events similar to those for mammalian GPCRs signal transduction. GPCRs are essential in many physiological processes associated with human diseases. Many aspects of structure and function are highly conserved across GPCRs, irrespective of primary amino acid sequence. This dissertation investigated the interactions of Ste2p with alpha-factor and Gpa1p. An overview of GPCRs in general with specific emphasis on Ste2p interactions with alpha-factor and Gpa1p are discussed in part I. Cross-linking studies of alpha-factor analogs containing 3,4-dihydroxylphenylalanine (DOPA) at positions 1 and 13 indicated that Trp1 and Tyr13 of [alpha]-factor are in close proximity to Lys269 and Cys59 of Ste2p, respectively when alpha-factor is bound to Ste2p. An alpha-factor synergist lacking the last two amino acids required for binding could only inhibit the cross-linking of DOPA at position 1 suggesting that the alpha-factor synergist interacts with the N-terminus of alpha-factor (described in part II). Part III describes the first report of an unnatural amino acid, p-benzoyl-L-phenylalanine (Bpa), replacement in a GPCR expressed in its native environment, and the use these receptors to photocapture a peptide ligand. Many of the Bpa-substituted Ste2p receptors exhibited biological activity and two of them, Phe55-Bpa and Tyr193-Bpa, were able to selectively capture alpha-factor in the ligand binding site after photoactivation; indicating that these residues may be in direct contact or in close proximity to alpha-factor when bound to Ste2p. Part IV reports for the first time the involvement of the third intracellular loop (IL3) in Ste2p homo-dimer formation, and also conformational changes at the cytoplasmic ends of TM5-TM6 of Ste2p induced by alpha-factor binding. Conformational changes in the C-terminus of Gpa1p occurring during Ste2p and Gpa1p activation are also discussed for the first time in part V. Variants of Ste2p-Gpa1p fusion proteins that displayed activities similar to Ste2p and Gpa1p are described in part VI. The final part of this dissertation discusses the overall conclusions and contains suggestions for future studies. The results obtained during this study should provide very important information about the mechanisms underlying the activation of GPCRs and G proteins.

Download or read book A Study of Interactions Between Saccharomyces Cerevisiae alpha factor and Its G Protein coupled Receptor Ste2p written by Byung-Kwon Lee and published by . This book was released on 2000 with total page 380 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Studies on the Role of Specific Residues of the Saccharomyces Cerevisiae alpha factor Pheromone Receptor Ste2p in the Inactive and Active State written by and published by . This book was released on 2005 with total page 192 pages. Available in PDF, EPUB and Kindle. Book excerpt: G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by seven-transmembrane (7TM) domains connected by intracellular and extracellular loops, an extracellular N-terminus, and an intracellular C-terminus. To date more than 1000 GPCRs have been identified, and these proteins recognize neurotransmitters, sensory molecules and chemotactic agents. These receptors are involved in the control of many aspects of metabolism and play important roles in diverse processes such as pain perception, growth and blood pressure regulation, and viral pathogenesis. Therefore, these proteins became important target for therapeutic agents and recent reports indicate that nearly 40% of drugs currently prescribed for human ailments target this family of proteins. The tridecapeptide [alpha]-factor pheromone (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) of Saccharomyces cerevisiae and Ste2p, its cognate GPCR, have been used extensively as a model for peptide ligand-GPCR structure and function. The power of yeast genetic was used to examine the structure-function relationship of [alpha]-factor receptor. Upon the [alpha]-factor binding to Ste2p, a signal is transduced via an associated guanine-nucleotide binding protein initiating a cascade of events that leads to the mating of haploid yeast cells. As only two GPCRs and two G proteins are encoded in the S. cerevisiae genome, S. cerevisiae provides an ideal system to study the relation between a peptide ligand and its GPCR in the absence of interfering biological complexities. Part I of this dissertation is an overview of the structure, receptor theory and conformational changes in receptor activation with specific emphasis on the peptide pheromone [alpha]-factor and its receptor Ste2p. Part II of this dissertation is a study of TM6 of Ste2p. Site-directed mutagenesis of a targeted portion of Ste2p TM6 was carried out. Among the Alanine substituted residues in the 262-270 region of Ste2p, only the Y266A mutant did not transduce signal yet exhibited only a small decrease in [alpha]-factor binding affinity. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted [alpha]-factor analogues (residues 1-4). Data from these studies suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of [alpha]-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding. Part III of this dissertation describes the specific interaction between residues N205 and Y266 of Ste2p in an active state not in resting state. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified N205 as a potential interacting partner with the Y266 residue. To test the interaction between N205 and Y266 residues of Ste2p, a series of biological and biochemical analysis coupled with mutation was carried out. First, a pH-dependent functional activity of N205H/Y266H double mutant suggests that the 205H and 266H residues interact in the activated state of the receptor. Second, the introduction of N205K or Y266D mutations into the P258L/S259L constitutively-active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/ P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor active state. Finally, we showed a di-sulfide formation only between N205C and Y266C in constitutively-active receptor not in WT receptor. Data from these studies suggest a specific interaction between N205 and Y266 in an active state, but not the resting state, of Ste2p. The final part of this dissertation reviews the overall conclusions and discussion. This part also contains suggestions for future experiments that could help to understand a conformation change during receptor activation. These studies should contribute to an understanding of the conformational differences between resting and active states of GPCRs.

Download or read book Structure function Studies of the Yeast Saccharomyces Cerevisiae alpha mating Factor Pheromone Receptor Ste2p written by and published by . This book was released on 2004 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: G protein-coupled receptors (GPCRs) are seven transmembrane domain cell surface proteins that respond to a variety of environmental cues. Response of these receptors to their cognate stimuli on the extracellular region of the cell results in a concurrent activation of a complex series of intracellular signaling pathways that prepare the cell for the required adjustments through regulation of gene expression levels. Participation of GPCRs in such intricate signal transduction pathways renders them important players in human diseases. The GPCR family of proteins therefore represents one of the largest classes of proteins to be targeted in the development of drug design for clinical applications. In light of the crucial role that GPCRs play in clinically important diseases, the focus of this dissertation has been on interactions between a GPCR and its ligand in a model eukaryotic organism, the budding yeast Saccharomyces cerevisiae. Very recently, the complete genome of the yeast S. cerevisiae has been sequenced. Detailed studies in this system along with the available sequence information have suggested a high conservation between the two eukaryotic organisms human and yeast. Therefore, the S. cerevisiae GPCR Ste2p and its associated pheromone ligand [alpha]-factor represent a good model system to study ligand-receptor interactions. The work presented in this dissertation describes results from a comprehensive mutagenesis approach on Ste2p aimed at determining residues of the receptor that are important in ligand binding and/or receptor activation. Regions of the receptor that have been the primary focus of the studies detailed in this dissertation are the first and third extracellular loops of Ste2p. Additional focus has been given to specific residues located in the transmembrane regions of Ste2p that have been predicted to interact with one another. Cys-scanning and Ala-scanning mutagenesis studies on the first extracellular loop, EL1, of Ste2p resulted in identification of a region of this loop harboring five functionally important residues that played an important role in the activation of the receptor but did not contribute to ligand binding. Structural studies on EL1 pointed to the possibility that this region of EL1 may attain a 310-helical structure in which the five functionally important residues may lie on one face of this helix. Collectively, all these studies underscored the important role of EL1 in Ste2p activation. Structure and function studies on the third extracellular loop, EL3, of Ste2p, using a Cys-scanning mutagenesis approach led to the identification of two additional residues that, upon mutation, resulted in a defective receptor.

Download or read book The N terminus of the Saccharomyces Cerevisiae G Protein coupled Receptor Ste2p written by Mohammad Seraj Uddin and published by . This book was released on 2013 with total page 205 pages. Available in PDF, EPUB and Kindle. Book excerpt: G protein-coupled receptors (GPCRs), the largest family of membrane proteins on the cell surface, play essential roles in signal transduction in all eukaryotic organisms. These proteins are responsible for sensing and detecting a wide range of extracellular stimuli and translating them to intracellular responses. This signaling requires a tight control for receptor activation without which abnormal signal leads to diseases. In fact, malfunctions of these receptors are associated with numerous pathological conditions and currently an estimated 40-50% of therapeutic drugs are designed to target these receptors suggesting that further increases in understanding of GPCRs and the signaling pathways they initiate will lead to new and more specific drug targets. We have used Saccharomyces cerevisiae GPCR Ste2p as a model system to understand structure-function relationships of these receptors. In this study, the role of the extracellular N-terminus has been examined using various biophysical methods with the anticipation to uncover its role in receptor function. It was found that some residues in the extracellular N-terminus were not accessible to a sulfhydryl reagent and that the alternating pattern of accessibility is consistent with the structure of a beta strand. This beta strand was found to be involved in dimer formation. Moreover, a conserved tyrosine residue in the middle of the beta strand was found to interact with two residues in the extracellular loop 1. It was also found that the N-terminus is involved in negative regulation and important for cell surface expression.

Download or read book Molecular Aspects of G Protein coupled Receptors written by Francisco Ciruela and published by Nova Publishers. This book was released on 2008 with total page 328 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the recent years, studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that intracellular domains of G protein-coupled receptors (GPCR) or heptaspanning membrane receptors (HSMRs) interact with intracellular proteins. These interactions are the basis of a protein network associated to these receptors which includes scaffolding proteins containing one or several PDZ (post-synaptic density-95, discs large, zona occludens-1) domains, signalling proteins and proteins of the cytoskeleton. The present book is focused on the emerging evidence for interactions of G protein-coupled receptors with scaffolding, cytoskeletal and signalling proteins that will play a role in the targeting, anchoring and functioning of these receptors in the plasma membrane, thus contributing to cell development and plasticity.

Download or read book Structural Characterization of the N terminal Region of the Saccharomyces Cerevisiae G protein Coupled Receptor Ste2p written by Stephanie Kendall and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Biosynthesis and Biophysical Analysis of Domains of the Alpha factor Receptor Ste2p written by Racha Estephan and published by . This book was released on 2005 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Pharmacological Profiling of G Protein coupled Receptors Using Saccharomyces Cerevisiae written by Gregory David Stewart and published by . This book was released on 2009 with total page 356 pages. Available in PDF, EPUB and Kindle. Book excerpt: It is commonly accepted that the nature of drug efficacy is not a linear concept, but rather a multi-dimensional signalling event. This gives rise to the potential for ligands to be functionally selective, which is preferential activation of a set of signalling cascades at the exclusion of others. This idea poses certain problems and provides new approaches for drug discovery programs with regard to choice of assay format and generation of pathway-selective compounds, respectively. In light of this, the majority of drug discovery programs that search for functionally selective ligands, combat this issue by using multiple endpoint assays, which vary depending on the targeted pathway. However, this approach is sometimes difficult to interpret for a number of reasons, such as the fact that many pathways are mediated by more than one upstream effector, the experimental conditions may vary between assay formats, for example, buffer composition and kinetics of activation. Moreover, using many functional endpoints is often expensive and inefficient. In this vein, this thesis discusses the development of a novel approach to screening for functional selective ligands, using a Saccharomyces cerevisiae expression system, which has been hitherto overlooked for this purpose. The ability of this system to detect functional selectivity of ligands within a G[alpha] protein family (G[alpha]i/o), and across multiple G[alpha] proteins (G[alpha]q, G[alpha]i1/2, G[alpha]12), was assessed. There was evidence that both orthosteric and allosteric ligands that bind M3 muscarinic acetylcholine receptors display functional selectivity, which was predicted using the yeast system; whilst the capability of the yeast system to predict selectivity between G[alpha]i/o subunits was comparatively reduced. In addition, the capacity of the yeast system to predict ligand parameters such as affinity and efficacy was also investigated. It was found that from data obtained in yeast, accurate affinity estimates could be generated. Furthermore, a potential use for yeast in estimating conformation-specific affinityvalues for agonists was revealed. Taken together, the evidence in this thesis suggests that the yeast signalling assays is a valuable and tractable platform for detecting pharmacological characteristics of existing and novel ligands.

Download or read book Toward an in Vivo G Protein Coupled Receptor GPCR Expression Interaction and Signaling Assay in Saccharomyces Cerevisiae written by Patrick McNeely and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The G Protein-Coupled Receptors (GPCRs) are membrane integral signaling proteins, which play a key role in signal transduction in myriad processes ranging from senses, organ development, chemotaxis, and neurotransmission in humans to cellular mating in yeasts. This diversity has lead to substantial interest in targeting these receptors for drug development. Native tissues typically express very little receptor, while the alternate hydrophilic and hydrophobic nature of the receptors, in conjunction with regulation of GPCRs in all cell types, conspire to present challenges to heterologous receptor expression for characterization. The present work is divided into two main subsections. The first introduces a promoter alteration to the expression of the human adenosine receptors in baker's yeast, Saccharomyces cerevisiae, and examines the resulting changes in expression of the receptor for the presence of properly folded and trafficked receptors at the plasma membrane. The second reports the incorporation of an easily monitored assay into the yeast genome for examination of the G protein signaling competence of the heterologous receptors expressed in our lab. Previous assays, particularly in yeast, have focused on ligand-binding competence of the receptor, though most reported crystal structures acknowledge the loss of signaling capacity of the resulting receptor, thus only partially filling the knowledge gap regarding the signaling behavior of these proteins in vivo.

Download or read book Formation of Multiple Dimer Interfaces In the Active and Inactive States of a Model G Protein coupled Receptor written by Hee Jung Kim and published by . This book was released on 2009 with total page 200 pages. Available in PDF, EPUB and Kindle. Book excerpt: G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by seven-transmembrane (7TM) domains connected by intracellular and extracellular loops, an extracellular N-terminus, and an intracellular Cterminus. GPCRs recognize neurotransmitters, sensory molecules and chemotactic agents and are involved in the control of many aspects of metabolism. Since GPCRs play important roles in diverse processes such as pain perception, growth and blood pressure regulation, and viral pathogenesis, GPCRs became important target for therapeutic agents. The tridecapeptide [alpha]-factor pheromone (W1H2W3L4Q5L6K--PG9Q10P11M12Y13) of Saccharomyces cerevisiae and Ste2p, its cognate GPCR, have been used extensively as a model for peptide ligand-GPCR structure and function. The power of yeast genetics has been used to examine the structure and function of Ste2p. Recently, GPCR homodimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. In this dissertation, Ste2p has been used to investigate GPCR dimerization. Part I of this dissertation is an overview of the GPCR structure and its ligandinduced conformational change with specific emphasis on the peptide pheromone [alpha]- factor and its receptor Ste2p. Part II of this dissertation is a study originally designed to probe inter-helical interaction between TM1 and TM7 of Ste2p. Site-directed mutagenesis and cysteine cross-linking with targeted residues of Ste2p were carried out. Although the anticipated inter-helical interactions were not identified from this study, the results provided strong evidence for Ste2p dimerization. Part III of this dissertation describes dimer interfaces including TM1 and TM7 of Ste2p. By using the disulfide cross-linking methodology, we studied the participation of specific residues at the intracellular boundary between TM1 and intracellular loop one and the entire TM7 in Ste2p dimerization. The final part of this dissertation contains a study of the participation of the Ste2p N-terminus in homo-dimer formation and the effect of ligand binding on this interaction. This part also includes overall conclusions and suggestions for future experiments that could contribute to an understanding of the dimer interfaces in Ste2p and the role of dimerization in the function of this receptor.

Download or read book Fungal Biotechnology and Bioengineering written by Abd El-Latif Hesham and published by Springer. This book was released on 2020-08-08 with total page 482 pages. Available in PDF, EPUB and Kindle. Book excerpt: Fungi are eukaryotic microorganisms that include both unicellular and multicellular species. They have a worldwide distribution and a wide range of applications in diverse sectors, from environmental, food and medicine to biotechnological innovations. Fungal biochemical genetics involves the study of the relationships between genome, proteome and metabolome, and the underlying molecular processes in both native and bioengineered fungi. This book provides a valuable resource on the challenges and potential of fungal biotechnology and related bioengineering and functional diversity for various industrial applications in the food, environmental, bioenergy and biorefining, and the biopharma sectors. In comparison to previous and related publications in the area of applied myco-biotech-engineering, this book bridges a knowledge gap in the areas related to prospects and investment as well as intellectual and technical issues. This book also provides information on recent commercial and economic interests in the area by juxtaposing the developments achieved in recent worldwide research and its many challenges.

Download or read book Regulation of Saccharomyces Cerevisiae Mating Pheromone Response written by Christopher Alvaro and published by . This book was released on 2015 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt: The yeast G-protein-coupled receptor (GPCR) Ste2 is an integral plasma membrane protein that initiates response to an extracellular stimulus (the peptide mating pheromone [alpha]-factor) by mediating ligand-dependent activation of a cognate heterotrimeric G-protein. Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest. In the research described in this dissertation, I found that three [alpha]-arrestins -- Rod1/Art4, Rog3/Art7 and Ldb19/Art1 -- serve as adaptors to promote the ubiquitinylation-dependent internalization of Ste2 and block its ability to signal, thereby desensitizing the cells to continued stimulation. Deleting the genes encoding these three [alpha]-arrestins increases the sensitivity of a MATa haploid cell to mating pheromone and results in an increase in Ste2 abundance at the plasma membrane. Conversely, overexpression of either Rod1 or Rog3 enhances the rate of adaptation. To contribute to negative regulation of the mating pheromone response pathway, Ldb19 requires binding of a HECT domain class of ubiquitin ligase, Rsp5, and most likely clears misfolded Ste2 from the PM. I found that Rod1 and Rog3 contribute to clearance of the pheromone-bound state of Ste2 and negatively regulate the mating pheromone response pathway by both Rsp5-dependent and Rsp5-independent mechanisms. In addition, I identified two classes of protein kinases (Snf1/AMPK and Ypk1/SGK) that phosphorylate and inactivate Rod1. Conversely, I showed that the phosphoprotein phosphatase responsible for dephosphorylating and re-activating Rod1 is calcineurin / PP2B. Because the S. cerevisiae genome does not encode any [beta]-arrestins, the findings I made and present in this dissertation are the first to show that [alpha]-arrestins alone are capable of negatively regulating a GPCR.

Download or read book G Protein coupled Receptors written by Tatsuya Haga, Ph.D. and published by CRC PressI Llc. This book was released on 2006 with total page 315 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a broad base of knowledge of G-protein-coupled receptors. Useful at both the university and industrial levels, this book is of particular interest to those who are developing therapeutic approaches to diseases using drugs that influence receptor activation.

Download or read book Screening Protein Ligand Interactions Using Microelectrode Arrays written by Sakshi Uppal and published by . This book was released on 2015 with total page 280 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Botrytis the Fungus the Pathogen and its Management in Agricultural Systems written by Sabine Fillinger and published by Springer. This book was released on 2015-12-16 with total page 488 pages. Available in PDF, EPUB and Kindle. Book excerpt: The fungal genus Botrytis is the focus of intensive scientific research worldwide. The complex interactions between this pathogen and the plants it infects and the economic importance of the diseases caused by Botrytis (principally grey mould) on more than 1400 species of cultivated plants pre- and post-harvest, render this pathogen of particular interest to farmers, advisers, students and researchers in many fields worldwide. This 20-chapter book is a comprehensive treatise covering the rapidly developing science of Botrytis and reflecting the major developments in studies of this fungus. It will serve as a source of general information for specialists in agriculture and horticulture, and also for students and scientists interested in the biology of this fascinating, multifaceted phytopathogenic fungal species.