Antagonistic and synergistic peptide analogs of the tridecapeptide mating pheromone of Saccharomyces cerevisiae

Eriotou-Bargiota, Effimia; Xue, Chu; Naider, Fred; Becker, Jeffrey M.

doi:10.1021/bi00117a036

Cited by 40 publications

(52 citation statements)

References 34 publications

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“…Similarly, binding of the G protein to the receptor is competed for by the presence of synthetic peptides that comprise a portion of the second loop or the C-terminal domain of rhodopsin (22,39). Many ␣-factor antagonists (14), including desTrp1,Ala3-␣-factor (our unpublished results), appear to elicit partial activation of the G protein, in that they show agonist activity when the target cells are supersensitive to ␣-factor as a result of the presence of the sst2 mutation. Since desTrp1,Ala3-␣-factor does not increase the exposure of the third intracellular loop, conformational changes at other sites in the ␣-factor receptor may contribute to signal transduction.…”

Section: Fig 4 Effects Of Mutations and Antagonist On The Cleavage mentioning

confidence: 78%

Agonist-Specific Conformational Changes in the Yeast α-Factor Pheromone Receptor

Bukusoglu

Jenness

1996

Molecular and Cellular Biology

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The yeast ␣-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, ␣-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-␣-factor, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. ␣-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor. The ␣-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants. Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes. These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor. The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.Receptors coupled to heterotrimeric GTP-binding proteins (G proteins) contain seven transmembrane helices and control cellular responses to a variety of stimuli, including hormones, neurotransmitters, light, and odorants (2, 13, 46). It is widely believed that the conformational changes induced by agonist binding permit these receptors to associate productively with G proteins, thus resulting in the exchange of GDP for GTP in the G ␣ subunit. Three lines of evidence suggest that the third intracellular loop of the receptor provides at least some of the intermolecular contacts that promote guanine nucleotide exchange: (i) work with chimeric receptors indicates that the third intracellular loop influences the specificity of G-protein interactions (13); (ii) mutations in the third intracellular loop (2,11,46,48) block coupling of receptors to their respective G proteins; and (iii) synthetic peptides that comprise the third loop bind G proteins in vitro, thereby stimulating nucleotide exchange and blocking the association of the G protein with the receptor (47). It has been proposed that structural constraints in receptors prevent the third intracellular loop from contacting the G protein when agonists are absent and that these structural constraints are relieved when the receptor assumes an agonist-activated conformation (36). However, there is as yet no direct evidence which demonstrates that increased exposure of the third intracellular loop is a specific consequence of agonist binding.The ability of ligands to promote changes in protein conformation has been largely pioneered by studying hemoglobin and allosteric enzymes (1, 37, 41). In general, alloster...

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Section: Fig 4 Effects Of Mutations and Antagonist On The Cleavage mentioning

confidence: 78%

Agonist-Specific Conformational Changes in the Yeast α-Factor Pheromone Receptor

Bukusoglu

Jenness

1996

Molecular and Cellular Biology

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“…Thus, binding of the tridecapeptide might disrupt and transform existing networks of interactions between various Ste2p residues to form a new network of interactions between the ␣-factor residues and Ste2p, resulting in the propagation of a conformational change across the receptor and G protein signaling. That residues at the N terminus of the pheromone are critical for receptor and G protein activation as shown by the fact that the deletion of the first two residues (Trp 1 and His 2 ) of ␣-factor results in a potent antagonist that binds well but cannot activate the receptor (22).…”

Section: Discussionmentioning

confidence: 99%

“…The tridecapeptide pheromone ␣-factor analogs [DOPA 1 ]␣-factor, and [Lys 7 (BioACA)]␣-factor were done as described previously (14,22).…”

Section: Synthesis and Characterization Of Dopa-␣-factor Analogsmentioning

confidence: 99%

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

Umanah

Huang

Ding

et al. 2010

Journal of Biological Chemistry

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G protein-coupled receptors (GPCRs)4 comprise the largest family of cell surface proteins present in the human genome responsible for communication between the internal and external environments in response to signal molecules, including hormones, pheromones, and neurotransmitters. GPCRs are characterized by the presence of seven membrane-spanning helical segments separated by alternating intracellular and extracellular loop regions (1-3). Despite their diverse ligands, all GPCRs perform similar functions, coupling the binding of ligands to the activation of specific heterotrimeric guanine nucleotide-binding proteins (G proteins), leading to the modulation of downstream effector proteins and molecules. Due to their involvement in a multitude of physiological functions, GPCRs are targeted by ϳ50% of the human drugs currently marketed (1,4,5). Detailed information about GPCR-ligand interactions is critical for our understanding of GPCR-ligand binding and function.Despite the differences in the binding mechanisms of various ligand groups in the GPCR family, there are some similarities that span this superfamily of proteins (1, 3, 6 -8). Ligand binding triggers conformational changes at the extracellular and transmembrane regions of the receptor, which are then propagated to the cytoplasmic surfaces, leading to G protein coupling and activation. Thus, ligand binding leads to the alteration of existing interhelical interactions, thereby promoting a set of new interactions that leads to a energetically favorable activated conformational state of the receptor (8, 9). Large ligands, such as proteins, bind to the extracellular loops of GPCRs, whereas small molecules like adrenergic agents bind within the transmembrane region of the receptor. Within the peptide-binding GPCRs, a combination of ligand binding to the extracellular loops followed by ligand penetra-

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“…Cells were incubated on ice for 30 min in the presence or absence of ␣-factor (WHWLQLKPGQPBY 12 ; where B indicates norleucine) or the des-Trp 1 des-His 2 antagonist (28) at a final concentration of 225 nM. Pheromones were synthesized and characterized as described previously (28,29 ]␣-factor, 225 nM final concentration) (30)were also tested along with the coumermycin antibiotic novobiocin (Sigma) at a final concentration of 2 M. The cells were warmed to room temperature and then supplemented with MTSEAbiotin at a final concentration of 0.1 mM. The MTSEA-biotin was prepared immediately before use as a 20 mM stock in dimethyl sulfoxide.…”

Section: Methodsmentioning

confidence: 99%

“…We chose to use whole cells for the SCAM analysis rather than isolated membranes to ensure that the topology of the receptor remained as close as possible to the intact native state. Accessibility was determined in both the presence and absence of ␣-factor or the des-Trp 1 des-His antagonist binds to WT Ste2p, but it does not initiate signal transduction (28). To prevent pheromone degradation and receptor internalization, the cells were maintained on ice during the 30-min pheromone incubation interval.…”

Section: Deglycosylation Of Signaling Compromised Mutants-wild-typementioning

confidence: 99%

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

Hauser

Kauffman

Lee

et al. 2007

Journal of Biological Chemistry

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In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the ␣-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr 101 through Gln 135 of EL1 in the presence and absence of the tridecapeptide ␣-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with ␣-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the ␣-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solventinaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding. G protein-coupled receptors (GPCRs)3 are ubiquitous in eukaryotes and have been found in a diversity of organisms ranging from yeast to humans. In most eukaryotes, GPCRs comprise 1-2% of the total genes in the genome (1) and participate in virtually all aspects of cellular physiology, including hormonal responses, neuronal transmission, and mediation of taste, smell, and vision (2). Modulation of GPCR function is a major pharmaceutical target, currently accounting for Ͼ30% of all drugs prescribed (3-5). Thus a thorough understanding of the nature of the receptor-ligand interaction and subsequent signal transduction is essential for the development of more effective and safer therapeutic agents.The structural hallmarks of this diverse collection of receptors are seven membrane-spanning domains linked by extracellular and intracellular loops, oriented such that the N terminus is external to the cell and the C terminus is internal. Although the structure-function relationships in the membrane-spanning domains and the intracellular loop regions have been well examined in many GPCRs (6 -11), few studies have focused on the importance of the extracellular loop structures and their role in receptor activation. In those studies where the extracellular domains were studied in deta...

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Antagonistic and synergistic peptide analogs of the tridecapeptide mating pheromone of Saccharomyces cerevisiae

Cited by 40 publications

References 34 publications

Agonist-Specific Conformational Changes in the Yeast α-Factor Pheromone Receptor

Agonist-Specific Conformational Changes in the Yeast α-Factor Pheromone Receptor

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

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