Probing the Binding Domain of the <i>Saccharomyces cerevisiae</i> α-Mating Factor Receptor with Fluorescent Ligands

Ding, Fa‐Xiang; Lee, B. K.; Hauser, Melinda; Davenport, Lesley; Becker, Jeffrey M.; Naider, Fred

doi:10.1021/bi0021535

Cited by 37 publications

(65 citation statements)

References 32 publications

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“…The model (Fig. 8) includes a well documented bend in the ␣-factor around the Pro 8 -Gly 9 sequence (36), with the Lys 7 side chain facing away from the TM domains and interacting with a binding pocket formed by the extracellular loops (37,38). Studies of the N terminus of ␣-factor indicated a strong preference for a large hydrophobic residue at the Nterminal position of the pheromone (39 -41); analyses of Alascanned ␣-factor analogs and fluorescent ␣-factor analogs indicated that the side chain of Trp 3 is in a hydrophobic pocket when bound to the receptor (14,42) (15).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…We found that Tyr 266 in TM6 plays an important role in the recognition of the N terminus of ␣-factor as well as switching of Ste2p into an activated state upon agonist binding (7), and using photo-affinity labeling we suggested that the N terminus of ␣-factor interacts with a region of the receptor spanning the upper portions of TM5 and TM6 and the connecting EL3 (8). Studies with ␣-factor analogs and fluorescently modified ␣-factor indicated that the binding environments of the position one (9) and position three side chains were hydrophobic, whereas the seventh residue was exposed to a partially hydrophilic environment suggesting that the middle portion of the pheromone was not buried in the transmembrane domains (10,11).…”

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confidence: 89%

Interacting Residues in an Activated State of a G Protein-coupled Receptor

Lee

Naider

Becker

2006

Journal of Biological Chemistry

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Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone ␣-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn 205 as a potential interacting partner with the Tyr 266 residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. 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. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.The tridecapeptide ␣-factor pheromone ( 1 WHWLQLKPGQPMY 13 ) of Saccharomyces cerevisiae and Ste2p, its cognate G protein-coupled receptor (GPCR), 3 have been used extensively as models for peptide ligand-GPCR structure and function (1). A major goal of GPCR studies is to ascertain the interactions between ligand and receptor to aid in the identification of analogs used for modulation of receptor activity and to understand how the receptor activates its signal transduction pathway. GPCRs are extremely important for medicine as they represent the target for the majority of prescribed drugs (2).To identify Ste2p residues or regions involved in ␣-factor binding, a number of experiments have been performed. Chimeric receptors between the closely related S. cerevisiae and Saccharomyces kluyveri ␣-factor receptors implicated the involvement of portions of EL1 (extracellular loop 1), EL3, and the N-terminal extracellular region of transmembrane 1 (TM1) in the specificity of ligand recognition (3, 4). Our group using site-directed mutagenesis of Ste2p and binding assays with different ␣-factor analogs suggested that portions of TM1 and TM6 were important for ligand interaction (5) and that the tenth residue of ␣-factor is in close proximity to Ser 47 and Thr 48 in TM1 of Ste2p (6). We found that Tyr 266 i...

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“…16,17 Among the many synthetic β-turn peptidomimetics restricted by cyclization, (R)-γ-lactam conformational constraint incorporating [3-(R)-amino-2-oxo-1-pyrrolidineacetamido] in place of ProGly at residues 8 and 9 was reported to have the best activity only equal to that of parent peptide, [Nle ing approach involving the photoactivatable groups attached to the α-factor backbone was developed. 28,30,44,[51][52][53]55,56 pBenzoyl-L-phenylalanine (Bpa) and 3,4-Dihydroxyphenylalanine (DOPA) was reported to have a desirable property for this purpose, although incorporation of these groups at various positions in α-factor have been shown to result in a less than 30-fold decrease in receptor affinity. 44,51,55 Biotin (Bio) tagging modification of DOPA analog (Bio 7…”

Section: 16mentioning

confidence: 99%

“…]α-factor) and 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) 30,53,54 developed for the detection of ligand-receptor conjugates were shown to reduce activity to about 33% that of parent α-factor. 44,56 Although the conjugation strategy of photoactivatable groups is not focused on enhancing potency, all of these analogs display receptor affinities varying from slightly better than that of α-factor to about 6-fold lower.…”

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Highly Active Analogs of α-Factor and Their Activities Against Saccharomyces cerevisiae

Ahn¹,

Hong²,

Jin³

et al. 2014

Bulletin of the Korean Chemical Society

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Thirteen analogs of tridecapeptide α-factor (WHWLQLKPGQPMY) of Saccharomyces cerevisiae with C-or N-terminal Trp extension and isosteric replacement by Aib at position 8 and 11, Trp at position 13, D-Ala at position 9, and Orn and Glu at position 6 were synthesized and assayed for their biological activity. Receptor binding assay was carried out using our newly developed spectrophotometric method with detector peptide 14. C-or N-terminal extended analogs, α-factor-[Trp] n (n =1-5) 1-5 and [N-Trp] 1 -α-factor 6, were all less active than native α-factor and gradual decreases in both activity and receptor affinity were observed with greater Trp extension. Trp-substituted analog at position 13, [Trp 13 ]α-factor 7, exhibited about 2-fold reductions in both activity and receptor affinity. Aib-substituted analogs, [Aib 8 ]α-factor 8 and [Aib 11]α-factor 9, showed 5-to 10-fold reduction in activity as well as 3-fold reduction in receptor affinity compared to native α-factor. [Orn 6]α-factor 10 demonstrated strong potency with a 7.0-fold increase in halo activity as well as 1.8-fold increase in receptor affinity compared to native α-factor. ]α-factor 11 exhibited 15-fold higher halo activity as well as nearly 3-fold higher receptor affinity compared to native α-factor.

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Probing the Binding Domain of the Saccharomyces cerevisiae α-Mating Factor Receptor with Fluorescent Ligands

Cited by 37 publications

References 32 publications

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

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

Interacting Residues in an Activated State of a G Protein-coupled Receptor

Highly Active Analogs of α-Factor and Their Activities Against Saccharomyces cerevisiae

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