scholarly journals Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

1993 ◽  
Vol 121 (4) ◽  
pp. 775-783 ◽  
Author(s):  
L A Jaffe ◽  
C J Gallo ◽  
R H Lee ◽  
Y K Ho ◽  
T L Jones
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Oocyte Maturation ◽  
Alpha Subunit ◽  
Meiotic Maturation ◽  
Amino Terminus ◽  
Hormone Response ◽  
Gamma Subunit ◽  
Gamma Subunits ◽  
Stimulation Of

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

scholarly journals Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

1993 ◽  
Vol 4 (10) ◽  
pp. 1027-1034 ◽  
Author(s):  
K Chiba ◽  
K Kontani ◽  
H Tadenuma ◽  
T Katada ◽  
M Hoshi
Keyword(s):  
G Protein ◽  
Oocyte Maturation ◽  
Kinase Activity ◽  
Germinal Vesicle ◽  
Bovine Brain ◽  
Cdc2 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Gamma Subunit ◽  
Gamma Subunits ◽  
Stimulation Of

beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.

scholarly journals The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein Gαt

1995 ◽  
Vol 311 (3) ◽  
pp. 987-993 ◽  
Author(s):  
R R Vaillancourt ◽  
N Dhanasekaran ◽  
A E Ruoho
Keyword(s):  
G Protein ◽  
Molecular Interactions ◽  
Molecular Mapping ◽  
Intramolecular Interactions ◽  
Alpha Subunit ◽  
Gamma Subunit ◽  
Terminal Domain ◽  
C Terminus ◽  
Gamma Subunits ◽  
Close Proximity

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to ‘tether’ [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the ‘acceptor’ domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2′-nitrophenylsulphenyl)-3-methyl-3′- bromoindolenine (‘BNPS-skatole’) and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

scholarly journals Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 791-799 ◽  
Author(s):  
Lisa M Mehlmann
Keyword(s):  
G Protein ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Follicle Cells ◽  
Meiotic Maturation ◽  
Meiotic Arrest ◽  
Epidermal Growth ◽  
High Level ◽  
G Protein Coupled ◽  
Stimulation Of

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Oocytes are arrested at the first meiotic prophase, held in meiotic arrest by the surrounding follicle cells until a surge of LH from the pituitary stimulates the immature oocyte to resume meiosis. Meiotic arrest depends on a high level of cAMP within the oocyte. This cAMP is generated by the oocyte, through the stimulation of the GsG-protein by the G-protein-coupled receptor, GPR3. Stimulation of meiotic maturation by LH occurs via its action on the surrounding somatic cells rather than on the oocyte itself. LH induces the expression of epidermal growth factor-like proteins in the mural granulosa cells that act on the cumulus cells to trigger oocyte maturation. The signaling pathway between the cumulus cells and the oocyte, however, remains unknown. This review focuses on recent studies highlighting the importance of the oocyte in producing cAMP to maintain arrest, and discusses possible targets at the level of the oocyte on which LH could act to stimulate meiotic resumption.

scholarly journals Structure-Function Studies of A. thaliana heterotrimeric G-protein subunits

2014 ◽  
Vol 70 (a1) ◽  
pp. C423-C423
Author(s):  
Sandra Quarantini ◽  
Hazal Kose ◽  
Burcu Kaplan-Turkoz ◽  
Anil Akturk ◽  
Zehra Sayers
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
G Proteins ◽  
Structural Parameters ◽  
Alpha Subunit ◽  
High Yield ◽  
E Coli ◽  
Functional Studies ◽  
Vis Spectroscopy ◽  
Gamma Subunits

Heterotrimeric G-proteins, consisting of alpha, beta, and gamma subunits, participate in major signaling pathways in eurkaryotes and mammals, including those in the nervous and sensory systems. G proteins are activated upon stimulation of G-protein coupled receptors and function as molecular switches through nucleotide cycling by the alpha subunit and by their interactions with intracellular effectors. The mechanism of activation and deactivation of the mammalian heterotrimer is well understood through extensive structural and functional studies, and is being used as a basis for predictions for mechanisms involving the plant heterotrimer. Recent evidence, however, indicates significant differences in the two systems [1]. We undertook a structural investigation of the A. thaliana complex in order to gain insights for G-protein activation and the molecular interactions in the G-protein related signaling pathways in plants. The alpha subunit GPA1 was cloned and expressed in P. pastoris and purified with high yield and homogeneity in functional form. Similarly the beta and gamma subunits (AGB1 and AGG2) were cloned and expressed in E. Coli. AGB1 expression resulted in production of unfolded protein whereas AGG2 could be obtained on its own with high yield and purity. Biochemical analyses and biophysical studies using SAXS, CD, DLS, UV-vis spectroscopy reveal structural parameters of GPA1 for comparison with its mammalian counterparts and indicate the propensity of GPA1 to form oligomers in solution. After purification form E. coli AGG2, can be stabilized in dimeric form and has a flexible and extended structure. Structure of the dimer is sensitive to the concentration of salt and reducing agent in the solution.

scholarly journals Localization of a heterotrimeric G protein gamma subunit to focal adhesions and associated stress fibers.

1994 ◽  
Vol 126 (3) ◽  
pp. 811-819 ◽  
Author(s):  
C A Hansen ◽  
A G Schroering ◽  
D J Carey ◽  
J D Robishaw
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Focal Adhesion ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Heterotrimeric G Proteins ◽  
Gamma Subunit ◽  
Surface Receptors ◽  
Gamma Subunits ◽  
Dual Staining

Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 alpha, 4 beta, and 5 gamma subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the gamma subunits. Using anti-peptide antibodies specific for each of the known gamma subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-gamma 5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the gamma 5 subunit with vinculin. In addition, the gamma 5 subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the gamma 5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the gamma 5 subunit and vinculin, suggesting that the association of the gamma 5 subunit with areas of focal adhesion was wide-spread.

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

Role of G proteins in stimulation of Na-H exchange by cell shrinkage

1992 ◽  
Vol 262 (2) ◽  
pp. C533-C536 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Transport Processes ◽  
Cell Shrinkage ◽  
Protein Activation ◽  
Kinase C ◽  
G Protein Activation ◽  
Barnacle Muscle Fibers ◽  
Stimulation Of

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.

scholarly journals Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins

1994 ◽  
Vol 297 (1) ◽  
pp. 189-193 ◽  
Author(s):  
A A Bominaar ◽  
P J M Van Haastert
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Wild Type ◽  
Inhibitory Pathway ◽  
Surface Receptors ◽  
G Protein Alpha Subunit ◽  
Camp Receptor ◽  
Protein Alpha Subunit

A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cyclic AMP (cAMP). This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild-type cells both cAMP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein alpha-subunit G alpha 2, showed no stimulation by either cAMP or GTP[S], indicating that G alpha 2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTP[S] was normal, suggesting that the defect in this mutant is upstream of the stimulatory G alpha 2. Inhibition of phospholipase C was achieved in wild-type cells by the partial antagonist 3′-deoxy-3′-aminoadenosine 3′,5′-phosphate (3′NH-cAMP). This inhibition was no longer observed in transformed cell lines lacking either the surface cAMP receptor cAR1 or the G-protein alpha-subunit G alpha 1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2.

scholarly journals Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation

2018 ◽  
Vol 293 (46) ◽  
pp. 17941-17952 ◽  
Author(s):  
Shawn K. Milano ◽  
Chenyue Wang ◽  
Jon W. Erickson ◽  
Richard A. Cerione ◽  
Sekar Ramachandran
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Binding Sites ◽  
Effector Proteins ◽  
Α Subunit ◽  
Gtp Binding ◽  
Effector Activity ◽  
Α Subunits ◽  
Phenylalanine Residue ◽  
Stimulation Of

Two regions on the α subunits of heterotrimeric GTP-binding proteins (G-proteins), the Switch II/α2 helix (which changes conformation upon GDP–GTP exchange) and the α3 helix, have been shown to contain the binding sites for their effector proteins. However, how the binding of Gα subunits to their effector proteins is translated into the stimulation of effector activity is still poorly understood. Here, we took advantage of a reconstituted rhodopsin-coupled phototransduction system to address this question and identified a distinct surface and an essential residue on the α subunit of the G-protein transducin (αT) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE). We started with a chimeric G-protein α subunit (αT*) comprising residues mainly from αT and a short stretch of residues from the Gi1 α subunit (αi1), which only weakly stimulates PDE activity. We then reinstated the αT residues by systematically replacing the corresponding αi1 residues within αT* with the aim of fully restoring PDE stimulatory activity. These experiments revealed that the αG/α4 loop and a phenylalanine residue at position 283 are essential for conferring the αT* subunit with full PDE stimulatory capability. We further demonstrated that this same region and amino acid within the α subunit of the Gs protein (αs) are necessary for full adenylyl cyclase activation. These findings highlight the importance of the αG/α4 loop and of an essential phenylalanine residue within this region on Gα subunits αT and αs as being pivotal for their selective and optimal stimulation of effector activity.

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