Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Brock, Carsten; Schaefer, Michael; Reusch, H. Peter; Czupalla, Cornelia; Michalke, Manuela; Spicher, Karsten; Schultz, Günter; Nürnberg, Bernd

doi:10.1083/jcb.200210115

Cited by 225 publications

(220 citation statements)

References 48 publications

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“…First, GTP-activated G s , G i , G q , or G o ␣ subunits were not able to stimulate the p101/p110␥ form of PtdIns 3-kinase. These experiments clarify earlier conflicting results on this point in the literature (9,10,42). As each ␣ subunit was purified from a column of immobilized ␤␥ dimers after activation with AlCl 3 and NaF, which induces a conformational change, the proteins are properly folded, active molecules.…”

Section: Discussionsupporting

confidence: 83%

“…The regulatory subunit for this isoform has a molecular mass of 101 kDa, has no recognizable protein-protein interaction domains, and dimerizes with the p110␥ catalytic moiety (2,9,10). The p110␥ form of the enzyme is highly expressed in cells of hematopoietic origin and is markedly activated by interaction with the G␤␥ dimer released after activation of receptors (9 -12).…”

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confidence: 99%

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Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Kerchner

Clay

McCleery

et al. 2004

Journal of Biological Chemistry

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The ability of G protein ␣ and ␤␥ subunits to activate the p110␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110␥ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTPactivated G s , G i , G q , or G o ␣ subunits were unable to activate PtdIns 3-kinase. Dimers containing G␤ 1-4 complexed with ␥ 2 -stimulated PtdIns 3-kinase activity about 26-fold with EC 50 values ranging from 4 to 7 nM. G␤ 5 ␥ 2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase C␤. A series of dimers with ␤ subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of ␤␥ with phosducin (␤1H311A␥2, ␤1R314A␥2, and ␤1W332A␥2) was tested, and only ␤1W332A␥2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the ␤ 1 subunit complexed with a panel of different G␥ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The ␤ 1 ␥ 2 , ␤ 1 ␥ 10 , ␤ 1 ␥ 12 , and ␤ 1 ␥ 13 dimers all activated PtdIns 3-kinase about 26-fold with 4 -25 nM EC 50 values. The ␤ 1 ␥ 11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110␥ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the ␥ subunit in determining the activation of PtdIns 3-kinase was examined using ␥ subunits with altered CAAX boxes directing the addition of farnesyl to the ␥ 2 subunit and geranylgeranyl to the ␥ 1 and ␥ 11 subunits. Replacement of the geranylgeranyl group of the ␥ 2 subunit with farnesyl inhibited the activity of ␤ 1 ␥ 2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the ␥ 1 and ␥ 11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all ␤ subunits, with the exception of ␤ 5 , interact equally well with PtdIns 3-kinase. In contrast, the composition of the ␥ subunit and its prenyl group markedly affects the ability of the ␤␥ dimer to stimulate PtdIns 3-kinase.The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 1 in the inner leaflet of the plasma membrane is critical to the regulation of cell function (1-3). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (1-3). In keeping with this central role, the level of PIP3 is tightly regulated, it can be elevated by multiple classes of receptors (1, 2), and there are specific phosphatidylinositol 5-phosphatases (SHIP and PTEN) that degrade the signal (4 -8).A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 ...

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Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Kerchner

Clay

McCleery

et al. 2004

Journal of Biological Chemistry

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“…Phosphatidylinositol 3-kinase ␥ (PI3K␥) plays a major role in cell migration toward chemokines by interacting with G␤␥ subunits (9,36). To follow PI3K␥ activity, we examined Akt phosphorylation, an event reflective of PI3K activation.…”

Section: G␤2 Sirna Treatment Modified the Kinetics Of Akt Phosphorylamentioning

confidence: 99%

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA

Hwang

Fraser

Choi

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking G␤2 but is unaffected in cells lacking G␤1, G␣i2, or G␣i3. Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by G␤2 knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking G␤1. Cells lacking G␤2 retain the rapid response but cannot sustain phosphoAkt1 levels. The phenotype of cells lacking G␤2 can be reversed by overexpression of either human G␤2 or mouse G␤1. These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, G␤2-derived G␤␥ is the most effective mediator of chemotaxis to C5a.

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“…FRET analysis was performed as described (28), using an inverted microscope Axiovert 100 equipped with a Plan-Apochromat ϫ63/1.4 objective (Carl Zeiss, Göttingen, Germany). In brief, CFP and YFP were alternately excited at 410 and 515 nm with a monochromator (Polychrome II; TILL Photonics, Grä felfing, Germany) in combination with a dual reflectivity dichroic mirror (Ͻ460 and 500 -520 nm; Chroma Technology, Rockingham, VT).…”

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confidence: 99%

Ligand-dependent Differences in the Internalization of Endothelin A and Endothelin B Receptor Heterodimers

Gregan

Jürgensen

Papsdorf

et al. 2004

Journal of Biological Chemistry

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117

106

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Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETEndothelins (ET-1, ET-2, and ET-3) 1 are important regulators in the vascular system. They act via two receptors: the endothelin A (ET A ) and endothelin B (ET B ) receptors (1, 2). Although human ET A and ET B receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ET A receptor stimulates G proteins of the G q/11 and G 12/13 families, the ET B receptor activates mainly G proteins of the G i and G q/11 families (3, 4). Whether ET B receptors also stimulate proteins of the G 12/13 family is still controversial and may depend on expression levels or cell types investigated (5, 6). Upon ligand binding, both receptor subtypes are rapidly desensitized by phosphorylation through the G protein-coupled receptor kinase type 2 (7). Following internalization via caveolae and/or clathrin-coated pits, the ET A receptor is recycled back to the cell surface (8, 9). In contrast, the ET B receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9, 10).The ET A receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca 2ϩ concentrations and activation of Rho proteins (11,12). The ET B receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13). In addition, ET A and ET B receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14 -16). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ET A and ET B receptors (17). Because atypical ligand binding was observed for cells co-expressing ET A and ET B receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15,16,18). For example, in epithelial cells of the anterior pituitary, ET B receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125 I-ET-1 binding only in the presence of the ET A receptor-selective antagonist BQ123 (16). In astrocytes, ET A and ET B receptors cooperatively control ET-1 clearance, because only the combi-

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Roles of Gβγ in membrane recruitment and activation of p110γ/p101 phosphoinositide 3-kinase γ

Cited by 225 publications

References 48 publications

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA

Ligand-dependent Differences in the Internalization of Endothelin A and Endothelin B Receptor Heterodimers

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