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G beta-gamma complex

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The G beta-gamma complex (Gβγ) is a tightly bound dimeric protein complex, composed of one Gβ and one Gγ subunit, and is a component of heterotrimeric G proteins. Heterotrimeric G proteins, also called guanine nucleotide-binding proteins, consist of three subunits, called alpha, beta, and gamma subunits, or Gα, Gβ, and Gγ. When a G protein-coupled receptor (GPCR) is activated, Gα dissociates from Gβγ, allowing both subunits to perform their respective downstream signaling effects. One of the major functions of Gβγ is the inhibition of the Gα subunit.[1]

This heterotrimeric G protein is illustrated with its theoretical lipid anchors. GDP is black. Alpha chain is yellow. Beta-gamma complex is blue. Membrane is Grey.
G-protein, β subunit
Identifiers
SymbolG-beta
InterProIPR016346
CATH2qns
SCOP22qns / SCOPe / SUPFAM
G-protein, γ subunit
Identifiers
SymbolG-gamma
PfamPF00631
InterProIPR036284
SMARTGGL
PROSITEPDOC01002
CATH2bcj
SCOP21gp2 / SCOPe / SUPFAM
OPM protein2bcj
CDDcd00068
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

History

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The individual subunits of the G protein complex were first identified in 1980 when the regulatory component of adenylate cyclase was successfully purified, yielding three polypeptides of different molecular weights.[2] Initially, it was thought that Gα, the largest subunit, was the major effector regulatory subunit, and that Gβγ was largely responsible for inactivating the Gα subunit and enhancing membrane binding.[1] However, downstream signalling effects of Gβγ were later discovered when the purified Gβγ complex was found to activate a cardiac muscarinic K+ channel.[3] Shortly after, the Gβγ complex associated with a mating factor receptor-coupled G protein in yeast was found to initiate a pheromone response.[4] Although these hypotheses were initially controversial, Gβγ has since been shown to directly regulate as many different protein targets as the Gα subunit.[1]

Recently, possible roles of the Gβγ complex in retinal rod photoreceptors have been investigated, with some evidence for the maintenance of Gα inactivation. However, these conclusions were drawn from in vitro experiments under unphysiological conditions, and the physiological role of the Gβγ complex in vision is still unclear. Nevertheless, recent in vivo findings demonstrate the necessity of the transducin Gβγ complex in the functioning of rod photoreceptors under low light conditions.[5]

Structure

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The Gβγ subunit is a dimer composed of two polypeptides, however it acts functionally as a monomer, as the individual subunits do not separate, and have not been found to function independently.[6]

The Gβ subunit is a member of the β-propeller family of proteins, which typically possess 4-8 antiparallel β-sheets arranged in the shape of a propeller.[7] Gβ contains a 7 bladed β-propeller, each blade arranged around a central axis and composed of 4 antiparallel β-sheets.[7] The amino acid sequence contains 7 WD repeat motifs of about 40 amino acids, each highly conserved and possessing the Trp-Asp dipeptide that gives the repeat its name.

The Gγ subunit is considerably smaller than Gβ, and is unstable on its own, requiring interaction with Gβ to fold, explaining the close association of the dimer. In the Gβγ dimer, the Gγ subunit wraps around the outside of Gβ, interacting through hydrophobic associations, and exhibits no tertiary interactions with itself. The N terminus helical domains of the two subunits form a coiled coil with one another that typically extends away from the core of the dimer.[7] To date, 5 β-subunit and 11 γ-subunit genes and have been identified in mammals.[6] The Gβ genes have very similar sequences, while significantly greater variation is seen in the Gγ genes, indicating that the functional specificity of the Gβγ dimer may be dependent on the type of Gγ subunit involved.[6] Of additional structural interest is the discovery of a so-called “hotspot” present on the surface of the Gβγ dimer; a specific site of the protein that binds to diverse range of peptides and is thought to be a contributing factor in the ability of Gβγ to interact with a wide variety of effectors.[8][9]

Synthesis and Modification

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Synthesis of the subunits occurs in the cytosol. Folding of the β-subunit is thought to be aided by the chaperone CCT (chaperonin containing tailless-complex polypeptide 1), which also prevents aggregation of folded subunits.[10] A second chaperone, PhLP (phosducin-like protein), binds to the CCT/Gβ complex, and is phosphorylated, allowing CCT to dissociate and Gγ to bind. Finally, PhLP is released, exposing the binding site for Gα, allowing for formation of the final trimer at the endoplasmic reticulum, where it is targeted to the plasma membrane.[11] Gγ subunits are known to be prenylated (covalently modified by the addition of lipid moieties) prior to addition to Gβ, which itself has not been found to be modified. This prenylation is thought to be involved in directing the interaction of the subunit both with membrane lipids and other proteins.[12]

Function

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The Gβγ complex is an essential element in the GPCR signaling cascade. It has two main states for which it performs different functions. When Gβγ is interacting with Gα it functions as a negative regulator. In the heterotrimer form, the Gβγ dimer increases the affinity of Gα for GDP, which causes the G protein to be in an inactive state.[13] For the Gα subunit to become active, the nucleotide exchange must be induced by the GPCR. Studies have shown that it is the Gβγ dimer that demonstrates specificity for the appropriate receptor and that the Gγ subunit actually enhances the interaction of the Gα subunit with the GPCR.[14][15] The GPCR is activated by an extracellular ligand and subsequently activates the G protein heterotrimer by causing a conformational change in the Gα subunit. This causes the replacement of GDP with GTP as well as the physical dissociation of the Gα and the Gβγ complex.[16]

Effector Signalling effect
GIRK2 activation
GIRK4 activation
N-type calcium channel inhibition
P/Q-type calcium channels inhibition
Phospholipase A activation
PLCβ1 activation
PLCβ2 activation
PLCβ3 activation
Adenylyl cyclase Type I, III, V, VI, VII inhibition
Adenylyl cyclase Type II, IV activation
PI3K inhibition
βARK1 activation
βARK2 activation
Raf-1 activation
Ras exchange factor activation
Bruton's tyrosine kinase activation
Tsk tyrosine kinase activation
ARF activation
Plasma membrane Ca2+ pump activation
p21-activated protein kinase inhibition
SNAP25 inhibition
P-Rex1 Rac GEF activation

[1]

Once separated, both Gα and Gβγ are free to participate in their own distinct signaling pathways. Gβγ does not go through any conformational changes when it dissociates from Gα and it acts as a signaling molecule as a dimer.[17] The Gβγ dimer has been found to interact with many different effector molecules by protein-protein interactions. Different combinations of the Gβ and Gγ subtypes can influence different effectors and work exclusively or synergistically with the Gα subunit.[1]

Gβγ signaling is diverse, inhibiting or activating many downstream events depending on its interaction with different effectors. Researchers have discovered that Gβγ regulates ion channels, such as G protein-gated inward rectifier channels,[3] as well as calcium channels.[18][9] In human PBMC, Gβγ complex has been shown to activate phosphorylation of ERK1/2.[19] Another example of Gβγ signaling is its effect of activating or inhibiting adenylyl cyclase leading to the intracellular increase or decrease of the secondary messenger cyclic AMP.[20] For more examples of Gβγ signaling see table. However, the full extent of Gβγ signaling has not yet been discovered.

Medical implications

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Drug design

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The Gβγ subunit plays a variety of roles in cell signalling processes and as such researchers are now examining its potential as a therapeutic drug target for the treatment of many medical conditions. However, it is recognized that there are a number of considerations to keep in mind when designing a drug which targets the Gβγ subunit:

  1. The Gβγ subunit is essential for the formation of heterotrimeric G protein through its association with the Gα subunit allowing the G proteins coupling to the GPCR. Therefore, any agent inhibiting the Gβγ subunits signalling effects must not interfere with the heterotrimeric G protein formation or Gα subunit signalling.
  2. Gβγ expression is universal throughout almost all the cells of the body so any agent acting to inhibit this subunit could elicit numerous side effects.
  3. Small molecule inhibitors that target the coupling of Gβγ to specific effectors and do not interfere with normal G protein cycling/ heterotrimeric formation, have the potential to work as therapeutic agents in treating some specific diseases.[17]

Targeting the Gβγ subunit in treatment

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Research has been conducted on how altering the actions of Gβγ subunits could be beneficial for the treatment of certain medical conditions. Gβγ signalling has been examined for its role in a variety of conditions including heart failure, inflammation and leukemia.[17][21]

Heart failure

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Heart failure can be characterized by a loss of β adrenergic receptor (βAR) signalling in heart cells.[22] When the βAR is stimulated by catecholamines such as adrenaline and noradrenaline, there is normally an increase in the contractility of the heart. However, in heart failure there are sustained and elevated levels of catecholamines which result in chronic desensitization of the βAR receptor. This leads to a decrease in the strength of heart contractions. Some research suggests that this chronic desensitization is due to the over activation of a kinase, G protein-coupled receptor kinase 2 (GRK2), which phosphorylates and deactivates certain G protein coupled receptors .[23] When the G protein coupled receptor is activated, the Gβγ subunit recruits GRK2 which then phosphorylates and desensitizes GPCRs like the βAR.[24] Preventing the interaction of the βγ subunit with GRK2 has therefore been studied as a potential target for increasing heart contractile function. The developed molecule GRK2ct is a protein inhibitor which inhibits the signalling properties of Gβγ subunit but does not interfere with alpha subunit signalling.[25] The over expression of GRK2ct has been shown to significantly rescue cardiac function in murine models of heart failure by blocking Gβγ subunit signalling.[26] In another study, biopsies were taken from patients with heart failure and virally induced overexpression of GRK2ct in the heart myocytes. Other tests showed an improvement in cardiac cell contractile function by inhibiting Gβγ.[27]

Inflammation

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When particular GPCRs are activated by their specific chemokines Gβγ directly activates PI3Kγ which is involved in the recruitment of neutrophils that contribute to inflammation.[28][29][30][31] It has been discovered that the inhibition of PI3Kγ significantly reduces inflammation.[28][29] PI3Kγ is the intended target molecule in the prevention of inflammation as it is the common signalling effector of many different chemokine and receptor types involved in promoting inflammation.[30][31] Although PI3Kγ is the intended target there are other isoforms of PI3 which perform different functions from PI3Kγ. Since PI3Kγ is specifically regulated by Gβγ, while other isoforms of PI3 are largely regulated by other molecules, inhibiting Gβγ signalling would provide the desired specificity of a therapeutic agent designed to treat inflammation.[17]

Leukemia

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The Gβγ subunit has been shown to activate a Rho guanine nucleotide exchange factor (RhoGef) gene PLEKHG2 which is upregulated in a number of leukemia cell lines and mouse models of leukemia.[32] Lymphocyte chemotaxis as a result of Rac and CDC42 activation as well as actin polymerization is believed to be regulated by the Gβγ activated RhoGef. Therefore, a drug inhibiting the Gβγ could play a role in the treatment of leukemia.[21]

References

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  1. ^ a b c d e Clapham DE, Neer EJ (1997). "G protein beta gamma subunits". Annual Review of Pharmacology and Toxicology. 37: 167–203. doi:10.1146/annurev.pharmtox.37.1.167. PMID 9131251.
  2. ^ Northup JK, Sternweis PC, Smigel MD, Schleifer LS, Ross EM, Gilman AG (November 1980). "Purification of the regulatory component of adenylate cyclase". Proceedings of the National Academy of Sciences of the United States of America. 77 (11): 6516–20. Bibcode:1980PNAS...77.6516N. doi:10.1073/pnas.77.11.6516. JSTOR 9587. PMC 350316. PMID 6935665.
  3. ^ a b Logothetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE (1987). "The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart". Nature. 325 (6102): 321–6. Bibcode:1987Natur.325..321L. doi:10.1038/325321a0. PMID 2433589. S2CID 4338529.
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  19. ^ Saroz, Yurii; Kho, Dan T.; Glass, Michelle; Graham, Euan Scott; Grimsey, Natasha Lillia (2019-10-19). "Cannabinoid Receptor 2 (CB 2 ) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes". ACS Pharmacology & Translational Science. 2 (6): 414–428. doi:10.1021/acsptsci.9b00049. ISSN 2575-9108. PMC 7088898. PMID 32259074.
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  21. ^ a b Runne C, Chen S (November 2013). "PLEKHG2 promotes heterotrimeric G protein βγ-stimulated lymphocyte migration via Rac and Cdc42 activation and actin polymerization". Molecular and Cellular Biology. 33 (21): 4294–307. doi:10.1128/MCB.00879-13. PMC 3811901. PMID 24001768.
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  30. ^ a b Stephens LR, Eguinoa A, Erdjument-Bromage H, Lui M, Cooke F, Coadwell J, Smrcka AS, Thelen M, Cadwallader K, Tempst P, Hawkins PT (April 1997). "The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101". Cell. 89 (1): 105–14. doi:10.1016/S0092-8674(00)80187-7. PMID 9094719. S2CID 16852661.
  31. ^ a b Stephens L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC, Hawkins PT (April 1994). "A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits". Cell. 77 (1): 83–93. doi:10.1016/0092-8674(94)90237-2. PMID 8156600. S2CID 53255676.
  32. ^ Ueda H, Nagae R, Kozawa M, Morishita R, Kimura S, Nagase T, Ohara O, Yoshida S, Asano T (January 2008). "Heterotrimeric G protein betagamma subunits stimulate FLJ00018, a guanine nucleotide exchange factor for Rac1 and Cdc42". The Journal of Biological Chemistry. 283 (4): 1946–53. doi:10.1074/jbc.M707037200. PMID 18045877.
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