scholarly journals Regulation of the G-protein-coupled alpha-factor pheromone receptor by phosphorylation.

1996 ◽  
Vol 16 (1) ◽  
pp. 247-257 ◽  
Author(s):  
Q Chen ◽  
J B Konopka
Keyword(s):  
G Protein ◽  
Yeast Cells ◽  
Special Role ◽  
Phosphorylation Sites ◽  
Pheromone Receptor ◽  
Protein Activation ◽  
C Terminus ◽  
G Protein Activation ◽  
Alpha Factor ◽  
Increased Sensitivity

The alpha-factor pheromone receptor activates a G protein signaling cascade that stimulates MATa yeast cells to undergo conjugation. The cytoplasmic C terminus of the receptor is not necessary for G protein activation but instead acts as a regulatory domain that promotes adaptation to alpha-factor. The role of phosphorylation in regulating the alpha-factor receptor was examined by mutating potential phosphorylation sites. Mutation of the four most distal serine and threonine residues in the receptor C terminus to alanine caused increased sensitivity to alpha-factor and a delay in recovering from a pulse of alpha-factor. 32PO4 labeling experiments demonstrated that the alanine substitution mutations decreased the in vivo phosphorylation of the receptor. Phosphorylation apparently alters the regulation of G protein activation, since neither receptor number nor affinity for ligand was significantly altered by mutation of the distal phosphorylation sites. Furthermore, mutation of the distal phosphorylation sites in a receptor mutant that fails to undergo ligand-stimulated endocytosis caused increased sensitivity to alpha-factor, which suggests that regulation by phosphorylation can occur at the cell surface and is independent of endocytosis. Mutation of the distal serine and threonine residues of the receptor also caused a slight defect in alpha-factor-induced morphogenesis, but the defect was not as severe as the morphogenesis defect caused by truncation of the cytoplasmic C terminus of the receptor. These distal residues in the C terminus play a special role in receptor regulation, since mutation of the next five adjacent serine and threonine residues to alanine did not affect the sensitivity to alpha-factor. Altogether, these results indicate that phosphorylation plays an important role in regulating alpha-factor receptor function.

scholarly journals Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

2020 ◽  
Vol 295 (51) ◽  
pp. 17486-17496
Author(s):  
Christopher T. Schafer ◽  
Anthony Shumate ◽  
David L. Farrens
Keyword(s):  
G Protein ◽  
Protein Activation ◽  
Canonical Class ◽  
Equilibrium Binding ◽  
C Terminus ◽  
G Protein Activation ◽  
Ligand Discovery ◽  
Pharmaceutical Agents ◽  
G Protein Coupled ◽  
Covalently Bound

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

scholarly journals Selective Interactions between Helix VIII of the Human μ-Opioid Receptors and the C Terminus of Periplakin Disrupt G Protein Activation

2003 ◽  
Vol 278 (35) ◽  
pp. 33400-33407 ◽  
Author(s):  
Giu-Jie Feng ◽  
Elaine Kellett ◽  
Carol A. Scorer ◽  
Jonathan Wilde ◽  
Julia H. White ◽  
...  
Keyword(s):  
G Protein ◽  
Opioid Receptors ◽  
Protein Activation ◽  
C Terminus ◽  
G Protein Activation ◽  
Μ Opioid Receptors ◽  
Selective Interactions

scholarly journals Direct Interactions with Gαi and Gβγ Mediate Nongenomic Signaling by Estrogen Receptor α

2007 ◽  
Vol 21 (6) ◽  
pp. 1370-1380 ◽  
Author(s):  
Premlata Kumar ◽  
Qian Wu ◽  
Ken L. Chambliss ◽  
Ivan S. Yuhanna ◽  
Susanne M. Mumby ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
G Protein ◽  
G Proteins ◽  
Cell Types ◽  
Estrogen Receptor Α ◽  
Erk Activation ◽  
Protein Activation ◽  
C Terminus ◽  
G Protein Activation ◽  
Oxide Synthase

Abstract Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor α (ERα). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ERα binds directly to Gαi and Gβγ. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251–260 and 271–595 of ERα, respectively. Studies of ERα complexed with heterotrimeric G proteins further show that estradiol causes the release of both Gαi and Gβγ without stimulating GTP binding to Gαi. Moreover, in COS-7 cells, the disruption of ERα-Gαi interaction by deletion mutagenesis of ERα or expression of blocking peptide, as well as Gβγ sequestration with β-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ERα-Gαi interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ERα mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.

scholarly journals Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

2000 ◽  
Vol 11 (3) ◽  
pp. 957-968 ◽  
Author(s):  
Carol L. Manahan ◽  
Madhavi Patnana ◽  
Kendall J. Blumer ◽  
Maurine E. Linder
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Yeast Cells ◽  
Pheromone Response ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Protein Activation ◽  
Pheromone Response Pathway ◽  
G Protein Activation

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among Gγsubunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the Gγsubunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of Gβγafter receptor-stimulated release from Gα. The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

scholarly journals β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

2021 ◽  
Vol 118 (11) ◽  
pp. e2017201118
Author(s):  
Keehun Kim ◽  
Shayla Paulekas ◽  
Fredrik Sadler ◽  
Tejas M. Gupte ◽  
Michael Ritt ◽  
...  
Keyword(s):  
G Protein ◽  
Dynamic Range ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Two Stage ◽  
Protein Activation ◽  
Intrinsic Efficacy ◽  
C Terminus ◽  
G Protein Activation ◽  
Structural Determinant

Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

Sequences in the Intracellular Loops of the Yeast Pheromone Receptor Ste2p Required for G Protein Activation†

Biochemistry ◽  
2003 ◽  
Vol 42 (10) ◽  
pp. 3004-3017 ◽  
Author(s):  
Andjelka Ćelić ◽  
Negin P. Martin ◽  
Cagdas D. Son ◽  
Jeffrey M. Becker ◽  
Fred Naider ◽  
...  
Keyword(s):  
G Protein ◽  
Pheromone Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Intracellular Loops

scholarly journals The C Terminus of the Saccharomyces cerevisiaeα-Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein

2000 ◽  
Vol 20 (14) ◽  
pp. 5321-5329 ◽  
Author(s):  
Mercedes Dosil ◽  
Kimberly A. Schandel ◽  
Ekta Gupta ◽  
Duane D. Jenness ◽  
James B. Konopka
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Protein Activation ◽  
Terminal Domain ◽  
G Protein Activation ◽  
Mutant Receptors ◽  
Signaling Activity

ABSTRACT Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway inMATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress theGPA1-A345T mutant Gα subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the α-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for α-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the α-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor–G-protein preactivation complexes.

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