Signalling through the leukotriene B4 receptor involves both αi and α16, but not αq or α11 G-protein subunits

Gaudreau, Rémi; Gouill, Christian Le; Métaoui, S; Lemire, S; Staňková, Jana; Rola‐Pleszczynski, Marek

doi:10.1042/bj3350015

Cited by 55 publications

(63 citation statements)

References 39 publications

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“…As reported by Gaudreau et al (20), COS-7 cells expressing both BLT1 and G␣ 16 show significantly higher IP accumulation than cells expressing BLT1 alone (Fig. 3A).…”

Section: Resultssupporting

confidence: 65%

“…We used HEK293 cells for GTP␥S binding assay, because HEK293 cells transiently expressing WT BLT1 showed higher LTB 4 -induced GTP␥S binding than COS-7 cells. We used COS-7 cells for the IP accumulation assay, since Gaudreu et al (20) had reported that COS-7 cells transfected with BLT1 and G␣ 16 showed LTB 4 -induced IP accumulation. CHO stable cells are commonly used for GPCR research, because they are effective in examining intracellular signaling and cellular events, such as calcium mobilization, cAMP assay, and chemotaxis.…”

Section: Discussionmentioning

confidence: 99%

“…Twenty-four hours later, culture medium was replaced with fresh medium containing 1 Ci/ml myo-[ 3 H]inositol (Amersham Biosciences), followed by incubation for another 18 -24 h. Cells were washed twice and preincubated for 10 min in Dulbecco's modified Eagle's medium containing 0.1% BSA and 10 mM LiCl, followed by stimulation with various concentrations of LTB 4 or vehicle for 30 min. Stimulation was terminated by replacing the medium with ice-cold 5% HClO 4 and incubating on ice for 2 h. Accumulated IP was purified by ion exchange chromatography (AG1-X8 resin; Bio-Rad), and radioactivity was measured by scintillation counting (20,21).…”

Section: Methodsmentioning

confidence: 99%

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Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Kuniyeda

Okuno

Terawaki

et al. 2007

Journal of Biological Chemistry

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. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTP␥S binding but intact inositol phosphate accumulation triggered by leukotriene B 4 stimulation. These results suggest that the i3-1 region is required only for G i activation. Moreover, in the i3-1 mutant, the deficiency in G i activation was accompanied by a loss of the high affinity leukotriene B 4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of G i proteins.Leukotriene B 4 (LTB 4 ) 3 is an inflammatory lipid mediator biosynthesized from membrane phospholipids by sequential enzymatic reactions (1-4). LTB 4 recruits and activates leukocytes, such as granulocytes, macrophages, monocytes, and T-cells, and plays pivotal roles in host defense and the pathogenesis of inflammatory and immune diseases (5, 6). These actions are mediated by two types of G-protein-coupled receptors (GPCRs). One is the LTB 4 receptor type-1 (BLT1), a high affinity LTB 4 receptor highly expressed in leukocytes (7). The other, BLT2, is a low affinity LTB 4 receptor expressed more ubiquitously than BLT1 in human tissues (8 -11).Many GPCRs can activate more than one G-protein subfamily member. For example, the prostaglandin E 2 receptor EP3D couples to G s and G o (12), platelet-activating factor receptor activates G i and G q (13,14), and the sphingosine 1-phosphate receptors S1P 1 and S1P 2 activate G i and G 12/13 (15). Previous reports suggest that in mediating different cellular events, BLT1 also utilizes different G-proteins, namely pertussis toxin (PTX)-sensitive G i/o and PTX-resistant G q subfamilies of G-proteins (7, 16). Promiscuous G-protein coupling enables GPCRs to mediate diverse signals.The molecular determinants of many receptors participating in G-protein coupling and activation have been investigated. With some exceptions, most of these studies emphasize the role of membrane-proximal regions of the second and third intracellular loops and of the cytoplasmic tail of the receptor (17). However, no consensus sequences for G-protein coupling have been clearly identified.We and others have reported that truncation of its cytoplasmic tail does not impair the ability of BLT1 to activate G-proteins (18,19). To identify regions of BLT1 responsible for G-protein coupling, we performed an extensive mutagenesis study of its intracellular domains. Here, we show that BLT1 distinguishes G i from G 16 at the amino terminus of the third intracellular region (the i3-1 region). Furthermore, Scatchard analyses of the i3-1 mutant revealed that BLT1 requires G i coupling to maintain a high affinity state for LTB 4 . A three-dimen-* Thi...

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“…As reported by Gaudreau et al (20), COS-7 cells expressing both BLT1 and G␣ 16 show significantly higher IP accumulation than cells expressing BLT1 alone (Fig. 3A).…”

Section: Resultssupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Kuniyeda

Okuno

Terawaki

et al. 2007

Journal of Biological Chemistry

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show abstract

“…A number of recent studies suggest that G␣ 16 coupling of chemoattractant and chemokine receptors may be important for leukocyte functions. It was shown that transmembrane signaling of the leukotriene B4 receptor is mediated by both G␣ i and G␣ 16 (43). In human T cells, alteration of the G␣ 16 expression level by cell-activating agents affects TCR-mediated functions (44).…”

Section: Discussionmentioning

confidence: 99%

Gα16 Couples Chemoattractant Receptors to NF-κB Activation

Yang

Sang

Rahman

et al. 2001

The Journal of Immunology

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The guanine nucleotide-binding regulatory protein α-subunit, Gα16, is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Gα16 coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a κB-driven luciferase reporter, Gα16, and the formyl peptide receptor responded to fMLP with a ∼7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IκBα and elevation of nuclear κB-DNA binding activity, indicating activation of NF-κB. In contrast to Gα16, expression of Gαq, Gα13, and Gαi2 resulted in a marginal increase in κB luciferase activity. A GTPase-deficient, constitutively active Gα16 mutant (Q212L) could replace agonist stimulation for activation of NF-κB. Furthermore, expression of Gα16 (Q212L) markedly enhanced TNF-α-induced κB reporter activity. The Gα16-mediated NF-κB activation was paralleled by an increase in phospholipase C-β activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca2+. The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCα enhanced the effect of Gα16, and a dominant negative PKCα partially blocked Gα16-mediated NF-κB activation. In addition to formyl peptide receptor, Gα16 also enhanced NF-κB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Gα16 in transcriptional regulation downstream of chemoattractant receptors.

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“…Stimulation was terminated by replacing the medium with ice-cold 5% HClO 4 and then incubating it on ice for 2 h. The accumulated IP was purified by ion exchange chromatography (AG1-X8 resin; Bio-Rad, Richmond, CA, USA) and eluted with 0.1 M formic acid/1.0 M ammonium formate for IP 3 elution. The radioactivity was measured by scintillation counting (Gaudreau et al, 1998;Fukunaga et al, 2001).…”

Section: Inositol Phosphate (Ip) Accumulation Assaymentioning

confidence: 99%

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

et al. 2009

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Signalling through the leukotriene B4 receptor involves both αi and α16, but not αq or α11 G-protein subunits

Cited by 55 publications

References 39 publications

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Gα16 Couples Chemoattractant Receptors to NF-κB Activation

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

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