Double-Mutant Cycle Scanning of the Interaction of a Peptide Ligand and Its G Protein-Coupled Receptor

Naider, Fred; Becker, Jeffrey M.; Lee, Yong–Hun; Horovitz, Amnon

doi:10.1021/bi602415u

Cited by 22 publications

(29 citation statements)

References 35 publications

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“…These substitutions had previously been reported to interact with bound ligand 15; 16; 22; 25 but were not recovered from our screen. However, each of these substitutions resulted in large decreases in the FL1/FL2 ratios of bound [Lys 7 (NBD),Nle 12 ]α-factor.…”

Section: Resultsmentioning

confidence: 96%

“…While Ste2p exhibits little sequence similarity to mammalian GPCRs, it is functionally interchangeable with some mammalian receptors 10; 11 . Several residues in the receptor that interact with ligand have been identified by chemical crosslinking and by characterizing amino acid substitutions in the ligand and receptor 12; 13; 14; 15; 16; 17; 18 . However, given the size of α-factor, it is likely that additional residues in Ste2p interact with bound peptide.…”

Section: Introductionmentioning

confidence: 99%

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Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

Mathew

Bajaj

Connelly

et al. 2011

Journal of Molecular Biology

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We describe a rapid method to probe for mutations in cell-surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence activated cell sorting to screen randomly-mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally-sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein coupled receptor, the procedure identified 22 substitutions that red-shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to directly contact the ligand. Instead, they alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the receptor’s activated conformation.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

Mathew

Bajaj

Connelly

et al. 2011

Journal of Molecular Biology

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“…6D) , and Leu 4 of the pheromone, suggesting that the N terminus of ␣-factor interacts cooperatively with residues at the TM5 and TM6 extracellular surface (43). 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.…”

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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“…9). Double mutant-cycle analyis (42) previously showed that Trp 1 and Trp 3 of α-factor interact strongly with N205 and Y266 residues that are located near the extracellular ends of TM5 and TM6, respectively (17, 52). Finally, the center of α-factor is thought to face toward the extracellular loop regions of Ste2p (17, 33).…”

Section: Discussionmentioning

confidence: 99%

Cross-Linking of a DOPA-Containing Peptide Ligand into Its G Protein-Coupled Receptor

et al. 2009

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The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine 7 ([Lys 7 (BioACA),-Nle 12 ,DOPA 13 ]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe 55 -Met 69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α-factor and residue Cys 59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor thereby defining a specific contact point between the bound ligand and its receptor.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins associated with signaling systems present in mammalians, plants, protozoans, fungi, and metazoans (1). Malfunctions of GPCRs contribute to diseases such as Alzheimer's, Parkinson's disease, diabetes, color blindness, asthma, depression, hypertension, stress, cardiovascular, and immune disorders (2,3). All GPCRs share a common structure consisting of seven transmembrane domains (TMDs) connected by three extracellular and three intracellular loops (1,4,5). Upon binding of their ligands, GPCRs undergo conformational changes that transduce NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 March 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the signal into the cell by activating a cascade of protein-protein interactions initiated through a heterotrimeric G protein complex (6).Ste2p, the Saccharomyces cerevisiae α-factor pheromone receptor, has been studied as a model for peptide-responsive GPCRs (7,8). Though Ste2p does not share recognizable sequence similarity with mammalian GPCRs or even with Ste3p, the a-factor pheromone receptor of yeast, all GPCRs have strong structural and functional similarities (9,10). For example, the packing and interactions between the fifth and sixth tr...

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Double-Mutant Cycle Scanning of the Interaction of a Peptide Ligand and Its G Protein-Coupled Receptor

Cited by 22 publications

References 35 publications

Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p

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

Cross-Linking of a DOPA-Containing Peptide Ligand into Its G Protein-Coupled Receptor

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