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

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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Structural basis of function in heterotrimeric G proteins

Quarterly Reviews of Biophysics ◽

10.1017/s0033583506004306 ◽

2006 ◽

Vol 39 (2) ◽

pp. 117-166 ◽

Cited By ~ 138

Author(s):

William M. Oldham ◽

Heidi E. Hamm

Keyword(s):

G Protein ◽

G Proteins ◽

Protein Complex ◽

Effector Proteins ◽

Protein Signaling ◽

Structural Determinants ◽

Α Subunit ◽

Protein Activation ◽

Downstream Effector ◽

G Protein Activation

1. Introduction 22. Heterotrimeric G-protein structure 32.1. G-protein α subunit 32.2. G-protein βγ dimer 82.3. Unique role of Gβ5 in complexes with RGS proteins 92.4. Heterotrimer structure 102.5. Lipid modifications direct membrane association 113. Receptor–G protein complex 113.1. Low affinity interactions between inactive receptors (R) and G proteins 113.2. Receptor activation exposes the high-affinity G-protein binding site 123.3. Receptor–G protein interface 143.4. Structural determinants of receptor–G protein specificity 153.5. Models of the receptor–G protein complex 173.6. Sequential interactions may form the receptor–G protein complex 194. Molecular basis for G-protein activation 194.1. Potential mechanisms of receptor-catalyzed GDP release 204.2. GTP-mediated alteration of the receptor–G protein complex 235. Activation of downstream effector proteins 245.1. Gα interactions with effectors 245.2. Gβγ interactions with effectors and regulatory proteins 266. G-protein inactivation 286.1. Intrinsic GTPase-activity of Gα 286.2. GTPase-activating proteins 307. Novel regulation of G-protein signaling 318. New approaches to study G-protein dynamics 328.1. Nuclear magnetic resonance spectroscopy 328.2. Site-directed labeling techniques 338.3. Mapping allosteric connectivity with computational approaches 348.4. Studies of G-protein function in living cells 369. Conclusions 3710. References 38Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein α subunit (Gα) binds GDP and Gβγ. Receptors activate G proteins by catalyzing GTP for GDP exchange on Gα, leading to a structural change in the Gα(GTP) and Gβγ subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Gα subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Gα, how these changes may lead to subunit dissociation and allow Gα and Gβγ to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor–G protein complex and how this interaction leads to GDP release from Gα. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor–G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588 ◽

Cited By ~ 23

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Abstract Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.bloodjournal753583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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Erratum

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1991.126 ◽

1991 ◽

Vol 11 (4) ◽

pp. 706-706

Keyword(s):

Blood Flow ◽

Cerebral Blood Flow ◽

Equilibrium State ◽

Rat Brain ◽

G Proteins ◽

Pertussis Toxin ◽

Adp Ribosylation ◽

Gtp Binding ◽

Reconstituted System ◽

Α Subunits

Ischemia of Rat Brain Decreases Pertussis Toxin-Catalyzed [32P] ADP Ribosylation of GTP-Binding Proteins (Gi1 and G0) in Membranes Katsunobu Takenaka, Yasunori Kanaho, Koh-ichi Nagata, Noboru Sakai, Hiromu Yamada, Yoshinori Nozawa [ Originally published in Journal of Cerebral Blood Flow and Metabolism 1991;11:155–160] On page 158 of the above, arrows were erroneously deleted from the equation in the following passage: Heterotrimers of G proteins that bind GDP to α subunits seem to be the preferred substrates for PTcatalyzed ADP ribosylation since guanine nucleotides (GDP and GTP) and 13'Y subunits stimulate ADP ribosylation in the reconstituted system and in membranes (Tsai et aI., 1984). These results indicate that the G proteins may exist at the equilibrium state as shown below: This omission was the result of a typesetting error, which the publisher regrets.

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Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

The Journal of Cell Biology ◽

10.1083/jcb.200808018 ◽

2008 ◽

Vol 183 (4) ◽

pp. 607-615 ◽

Cited By ~ 129

Author(s):

Rita Sinka ◽

Alison K. Gillingham ◽

Vangelis Kondylis ◽

Sean Munro

Keyword(s):

Drosophila Melanogaster ◽

Golgi Apparatus ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Coiled Coil ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Rab Family ◽

First Contact

Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain golgins, whose C termini bind the Arf-like 1 G protein on the trans-Golgi, can also bind four members of the Rab family of G proteins. The Rab2-, Rab6-, Rab19-, and Rab30-binding sites are within the coiled-coil regions that are not required for Golgi targeting. Binding sites for two of these Rabs are also present on two coiled-coil proteins of the cis-Golgi, the Drosophila melanogaster orthologues of GM130 and GMAP-210. We suggest an integrated model for a tentacular Golgi in which coiled-coil proteins surround the Golgi to capture and retain Rab-containing membranes, excluding other structures such as ribosomes. Binding sites for diverse Rabs could ensure that incoming carriers are captured on first contact and moved to their correct destination within the stack.

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New thoughts on the role of the βγ subunit in G protein signal transduction

Biochemistry and Cell Biology ◽

10.1139/o00-075 ◽

2000 ◽

Vol 78 (5) ◽

pp. 537-550 ◽

Cited By ~ 1

Author(s):

Barbara Vanderbeld ◽

Gregory M Kelly

Keyword(s):

Signal Transduction ◽

G Protein ◽

G Proteins ◽

G Protein Coupled Receptors ◽

Limited Information ◽

Α Subunit ◽

Downstream Effectors ◽

Receptor Signal ◽

G Protein Coupled

Heterotrimeric G proteins are involved in numerous biological processes, where they mediate signal transduction from agonist-bound G-protein-coupled receptors to a variety of intracellular effector molecules and ion channels. G proteins consist of two signaling moieties: a GTP-bound α subunit and a βγ heterodimer. The βγ dimer, recently credited as a significant modulator of G-protein-mediated cellular responses, is postulated to be a major determinant of signaling fidelity between G-protein-coupled receptors and downstream effectors. In this review we have focused on the role of βγ signaling and have included examples to demonstrate the heterogeneity in the heterodimer composition and its implications in signaling fidelity. We also present an overview of some of the effectors regulated by βγ and draw attention to the fact that, although G proteins and their associated receptors play an instrumental role in development, there is rather limited information on βγ signaling in embryogenesis.Key words: G protein, βγ subunit, G-protein-coupled receptor, signal transduction, adenylyl cyclase.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.2.c533 ◽

1992 ◽

Vol 262 (2) ◽

pp. C533-C536 ◽

Cited By ~ 41

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.121.4.775 ◽

1993 ◽

Vol 121 (4) ◽

pp. 775-783 ◽

Cited By ~ 68

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.

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Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels

Biochemical Journal ◽

10.1042/bj20011438 ◽

2002 ◽

Vol 364 (2) ◽

pp. 369-376 ◽

Cited By ~ 18

Author(s):

Pavel FLACHS ◽

JiŘí NOVOTNÝ ◽

Filip BAUMRUK ◽

Kristina BARDOVÁ ◽

Lenka BOUŘOVÁ ◽

...

Keyword(s):

Adipose Tissue ◽

Transgenic Mice ◽

G Protein ◽

Energy Status ◽

Α Subunit ◽

Fat Depots ◽

Ucp1 Expression ◽

White Fat ◽

Stimulation Of

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein α subunit was increased while that of the inhibitory G-protein α subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.

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Is Receptor Cross-Regulation in Human Heart Caused by Alterations in Cardiac Guanine Nucleotide-Binding Proteins?

Clinical Science ◽

10.1042/cs0850393 ◽

1993 ◽

Vol 85 (4) ◽

pp. 393-399 ◽

Cited By ~ 4

Author(s):

A. Ferro ◽

C. Plumpton ◽

M. J. Brown

Keyword(s):

G Protein ◽

G Proteins ◽

Atrial Appendage ◽

Right Atrial ◽

Α Subunit ◽

Guanine Nucleotide Binding ◽

Quantitative Changes ◽

Right Atrial Appendage ◽

Nucleotide Binding Proteins ◽

Guanine Nucleotide Binding Proteins

1. Guanine nucleotide-binding proteins (G-proteins) play a central role in signal transduction between a wide variety of cell-surface receptors and intracellular second messenger systems. Recently, we and others have demonstrated that cross-regulation can occur between a variety of G-protein-linked receptors in human heart. Chronic β1-adrenoceptor blockade gives rise to sensitization of β2-adrenoceptor and of 5HT4-receptor responses, both of which are mediated via stimulation of adenylate cyclase through stimulatory G-proteins (Gs), and also gives rise to desensit-ization of muscarinic M2-receptor responses, which inhibit adenylate cyclase through inhibitory G-proteins (Gi). 2. In order to investigate whether these effects are due to quantitative changes in cardiac G-protein isoforms, we measured their abundance in right atrial appendage from patients taking or not taking β1-adrenoceptor antagonists, by immunoblotting. 3. Samples of right atrial appendage homogenate were subjected to SDS/PAGE, and proteins were electroblotted on to nitrocellulose membranes. These were then probed with specific anti-G protein anti-sera, and binding was revealed by means of a secondary antibody labelled with alkaline phosphatase and using a chromogenic substrate. The resulting bands were quantified by laser densitometry. 4. No quantitative differences were detected, between these two groups of patients, in the amounts of α-subunit of ‘long’ or ‘short’ Gs isoforms (GsαL and GsαS), or in the amounts of Gi 1 + 2 α-subunit (Giα1 + 2). Nor was any difference found in the abundance of the β-subunit of G-proteins. No ‘other’ G-protein (Go) was detectable in these samples by immunoblotting. 5. We conclude that the phenomenon of receptor cross-regulation which we have previously observed in human right atrial appendage is unlikely to be explained by quantitative changes at the G-protein level.

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