Receptor-Mediated Activation of Heterotrimeric G-Proteins: Current Structural Insights

Johnston, Christopher A.; Siderovski, David P.

doi:10.1124/mol.107.034348

Cited by 129 publications

(127 citation statements)

References 108 publications

(173 reference statements)

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“…Our in silico results predicted that Phe 336 is the most energetically important residue both in maintaining the basal state and in promoting the receptor-bound conformation (6). Our proposed mechanism involves Phe 336 acting as a relay to transmit conformational changes via strands ␤2 and ␤3 and helix ␣1 to the phosphate-binding loop (5,6). These studies are supported by recently published computational studies (11,19,20).…”

supporting

confidence: 74%

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Kaya¹,

Lokits

Gilbert³

et al. 2016

Journal of Biological Chemistry

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G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the ␣5 helix of G␣ i1 , especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in G␣ i1 by inserting 4 alanine amino acids into the ␣5 helix between residues Gln 333 and Phe 334 (Ins4A). This extends the length of the ␣5 helix without disturbing the ␤6-␣5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His 57 , Phe 189 , Phe 191 , and Phe 336 ; it also disturbs the rotation of the ␣5 helix and the -interaction between His 57 and Phe 189 . In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between ␣5, ␣1, and ␤2-␤3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the ␣5 helix for G protein release from the receptor after GTP binding.Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, act as a molecular switches that turn on intracellular signaling cascades in response to the activation of G protein-coupled receptors by extracellular stimuli. Therefore, G proteins have a critical role in many different cellular responses (1-6).The G␣ subunit binds GDP and forms a tight complex with the G␤␥ subunits. Activated G protein-coupled receptors can catalyze the exchange of GDP for GTP, which leads to the dissociation of the receptor-G protein complex into isolated receptor and G␣ and G␤␥ subunits. Both the G␣ and G␤␥ subunits can then stimulate or inhibit downstream effectors. Signal propagation ceases after the G␣ subunit hydrolyzes GTP, returns to the inactive state, and rebinds to the G␤␥ subunit, regenerating the GDP-bound heterotrimeric state.Previous studies showed that the activated receptor directly interacts with the G protein by binding to the C-terminal ␣5 helix of G␣, inducing a rigid body rotation and translation that pull this helix into a hydrophobic pocket on the receptor (7,8). This leads to the rearrangement of the interfaces between helices ␣5, ␣1, and the ␤2-␤3 strands and between ␣5 and the ␤6-␣5 loop (1, 7, 9 -11). Residue Phe 336 in the ␣5 helix is highly conserved in small (12, 13) and large GTPases (14) in both the animal and plant kingdoms (15-18). Our in silico results predicted that Phe 336 is the most energetically important residue both in maintaining the basal state and in promoting the receptor-bound conformation (6...

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supporting

confidence: 74%

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Kaya¹,

Lokits

Gilbert³

et al. 2016

Journal of Biological Chemistry

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“…Bovine and human ␥ 2 have identical amino acid sequences, and mouse ␣ q differs from human ␣ q in but one residue (S141A). There are several indications that this residue has no role in ␣ q function: (i) In mouse ␣ q , Ser 141 is in a long loop connecting helices ␣E and ␣F of the helical domain (PDB accession codes 2BCJ and 2RGN); it does not reside in any of the three switch domains or in regions known to interact with PLC␤ isozymes and is not among the residues that contact the bound guanine nucleotide and G␤␥ (38,39). (ii) Secondary structure prediction algorithms do not predict a structural difference between mouse and human ␣ q ␣E-loop-␣F region (40, …”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Gutman

Walliser

Piechulek

et al. 2010

Journal of Biological Chemistry

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We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-␤ 2 (PLC␤ 2 ) by G protein ␣ q and ␤␥ dimers. We show that activation by ␣ q and ␤␥ differ from activation by Rac2 and from each other. Stimulation by ␣ q enhanced the plasma membrane association of PLC␤ 2 , but not of PLC␤ 2 ⌬, which lacks the ␣ q -interacting region. Although ␣ q resembled Rac2 in increasing the contribution of exchange to the FRAP of PLC␤ 2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLC␤ 2 by ␣ q occurred by enhancing PLC␤ 2 association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by ␤␥ shifted the FRAP of PLC␤ 2 and PLC␤ 2 ⌬ to pure lateral diffusion 3-to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLC␤ 2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) in localized versus dispersed populations. PLC␤ 2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLC␤ 2 to act locally on PtdInsP 2 at specific domains. Activation by ␣ q leads to lipid-like diffusion of PLC␤ 2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by ␤␥ recruits PLC␤ 2 to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP 2 populations. Phospholipase C-␤ (PLC␤)4 isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) to produce inositol 1,4,5-trisphosphate and diacylglycerol (1-3). They are activated to different extents by heterotrimeric G protein ␣ q subunits (␣ q ) and ␤␥ dimers (␤␥) (1-3). PLC␤ 2 is also activated by the Rho GTPases Rac and Cdc42 (4 -7). Activation by Rac has also been demonstrated for PLC␥ 2 (8). PLC␤ 2 has long been known to be expressed in hematopoietic cells (2, 3) but is also encountered in a variety of other cell types and tissues, including smooth muscle cells (9) and several brain regions (10, 11). Moreover, PLC␤ 2 was shown to be essential for taste perception via certain G protein-coupled oral taste receptors (12).Activation of PLC␤ 2 by ␣ q and related ␣ subunits requires the C-terminal region of the enzyme; mutants with deletions in this region (e.g. PLC␤ 2 ⌬, that lacks the Phe 819 -Glu 1166 segment) are resistant to stimulation by ␣ q , but undergo activation by ␤␥ and Rac/Cdc42 (7, 13-15). Recent results show that ␤␥ and Rac/Cdc42 activate PLC␤ 2 by interacting, at least in part, with different regions of the effector enzyme. Thus, although the pleckstrin homology (PH) domain of PLC␤ 2 is dispensable for activation of the enzyme by...

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“…GTP then replaces GDP and a new structural change occurs, which causes the detachment of one subunit (the socalled G␣ subunit) from the other two. The G␣ subunit binds and activates various enzymes and effectors (Kristiansen, 2004;Johnston and Siderovski, 2007). In the case of ORs, it activates AC type III.…”

Section: Structural Prediction and MD Studiesmentioning

confidence: 99%

Computational molecular biology approaches to ligand‐target interactions

et al. 2009

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Binding of small molecules to their targets triggers complex pathways. Computational approaches are keys for predictions of the molecular events involved in such cascades. Here we review current efforts at characterizing the molecular determinants in the largest membrane-bound receptor family, the G-protein-coupled receptors "GPCRs…. We focus on odorant receptors, which constitute more than half GPCRs. The work presented in this review uncovers structural and energetic aspects of components of the cellular cascade. Finally, a computational approach in the context of radioactive boron-based antitumoral therapies is briefly described.

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Receptor-Mediated Activation of Heterotrimeric G-Proteins: Current Structural Insights

Cited by 129 publications

References 108 publications

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Computational molecular biology approaches to ligand‐target interactions

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