A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Lin, Jennifer; Duell, Ken; Konopka, James B.

doi:10.1128/mcb.24.5.2041-2051.2004

Cited by 28 publications

(59 citation statements)

References 59 publications

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“…The drop in reactivity is thought to correspond to the membrane interface region of the TMDs. Although a number of different factors can influence the reactivity of any one particular residue in STE2, a drop in reactivity over 3-5 residues is consistent with expectations for an α-helical TMD that is crossing the membrane boundary zone (14). Similar results were observed for the solvent accessibility of Cys-substituted residues in rhodopsin (28)(29)(30)(31), and the results were subsequently found to correlate well with the TMD arrangement observed in the rhodopsin crystal structure (27).…”

Section: Ste2 Topologysupporting

confidence: 81%

“…The degree of biotinylation was then determined by quantitative Western blot analysis using anti-HA antibody to detect total STE2, and Streptavidin to detect biotinylated STE2 as described below. This procedure is a modification of the method used previously to analyze the reactivity with MTSEA-biotin of residues in the extracellular regions of STE2 (14). Previously, Streptavidin beads were used to precipitate the biotinylated STE2 proteins, whereas the new approach uses anti-HA beads.…”

Section: Mtsea-biotin Reactionsmentioning

confidence: 99%

“…Cys substitution mutations were introduced into the residues that span across the intracellular loops and the cytoplasmic end of TMD7 so that their solvent accessibility could be probed with MTSEA-biotin, a thiol reactive probe that does not react with membrane-buried residues (12). A similar approach used to define that topology of the extracellular regions of Ste2 demonstrated that the extracellular ends of the TMDs play a key role in ligand binding and in promoting the active receptor conformation (13,14), and also provided a structural framework for comparing Ste2 to mammalian GPCRs (5). Therefore, the accessibility of residues in the cytoplasmic regions of Ste2 was assayed to provide a structural framework for understanding the role of residues that function in G protein activation.…”

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confidence: 99%

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Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Choi

Konopka

2006

Biochemistry

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The yeast α-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 were substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEAbiotin (2-([biotinoyl] amino) ethyl methanethiosulfonate), which reacts with solvent accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH ≥7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gproteincoupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1,2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for † This work was supported by National Institutes of Health Grant GM55107 awarded to J.B.K. post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying ...

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Section: Ste2 Topologysupporting

confidence: 81%

Section: Mtsea-biotin Reactionsmentioning

confidence: 99%

mentioning

confidence: 99%

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Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Choi

Konopka

2006

Biochemistry

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“…Induced conformational changes in the ECL2 have been documented in several GPCRs, including the D2 dopamine receptor and C5A complement receptor (44,45) upon ligand occupancy of the orthosteric pocket in the TM domain. Long range conformational changes associated with ligand binding have been shown in ECL1 of glucagon receptor (46); the N terminus, ECL1, and extracellular ends of all TM helices of Saccharomyces cerevisiae G protein-coupled receptor Ste2p are known (47)(48)(49). These observations suggest that a mutation in a particular domain of the receptor causes a local structural perturbation that is eventually transmitted to other domains, leading to a global consequence in terms of affinity toward ligands and G protein.…”

Section: Ecl2 Conformation In Activation State Mutants Of At1rmentioning

confidence: 89%

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

Ünal

Jagannathan

Bhatnagar

et al. 2013

Journal of Biological Chemistry

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Background:The binding of ligands to the orthosteric pocket induces ligand-specific conformational changes all through domains of G protein-coupled receptors. Results: Loss of function and gain of function mutations in the transmembrane domain spontaneously induce changes similar to a ligand-specific conformation in the extracellular domain. Conclusion: Coupling between domains determines the overall signaling state of GPCRs. Significance: Targeting domain interfaces might be the future for GPCR-specific drug development.

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“…These studies together with extensive molecular biology investigations (19)(20)(21)(22)(23) have indicated likely interactions between Y266 and residues near the amine terminus of α-factor. Very recently, biochemical evidence based on disulfide crosslinking indicated that N205 and Y266 might be close in an activated state of Ste2p as represented by a constitutively active mutant of this GPCR (24).…”

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confidence: 99%

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

et al. 2007

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The interaction between the yeast G protein coupled receptor (GPCR), Ste2p, and its α-factor tridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwise interaction energy of each ligand residue with two receptor residues, N205 and Y266, was determined. The mutations N205A and Y266A were previously shown to result in deficient signaling but cause only a 2.5-fold and 6-fold decrease, respectively, in the affinity for α-factor. The analysis shows that residues at the amine terminus of α-factor interact strongly with N205 and Y266 whereas residues in the center and at the carboxyl terminus of the peptide interact only weakly if at all with these receptor residues. Multiple-mutant thermodynamic cycle analysis was used to assess whether the energies of selected pairwise interactions between residues of the α-factor peptide changed upon binding to Ste2p. Strong positive cooperativity between residues 1 through 4 of α-factor was observed during receptor binding. In contrast, no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminus of α-factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutant cycle analyses of the binding of an alaninescanned peptide to wild-type and mutant GPCRs can provide detailed information on contributions of inter-and intra-molecular interactions to the binding energy and potentially prove useful in developing 3D models of ligand docked to its receptor.Determination of contacts between peptide ligands and their cognate G protein-coupled receptors (GPCRs) 2 and the respective energetics of these interactions is essential for understanding how binding is transduced to intracellular signaling. Many studies have employed structure-activity relationships involving ligand analogs and mutagenesis of receptors to discern receptor-ligand interactions (reviewed in refs.1,2). Complementary investigations have used photocrosslinking to provide biochemical evidence for contacts (3,4). In the 1980s, Fersht and coworkers and Horovitz introduced double-mutant cycle analysis to provide thermodynamic evidence for the interaction between groups within one protein or in ligand-protein complexes (5-7). The method has been applied to a number of receptor systems including the ligand-gated ion channel nicotinic (8,9) and the GPCR muscarinic (10) acetylcholine receptors. The concept of this method is illustrated in a cycle for a ligand binding to its GPCR ( Figure 1A). If the effect on the binding free energy (or the free energy of some other process) of the double mutation is not equal to the sum of effects of the single mutations then the two residues are coupled. Non-zero pairwise coupling energies calculated from such † This work was supported by research grants GM22086 and GM22087 from the National Institutes of Health. cycles reflect interactions that can be either direct or indirect. Coupling energies found for two directly interacting residues can be converted to a distance constrain...

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A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Cited by 28 publications

References 59 publications

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

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

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