Abstract
Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a Gα i1-[γ- 32P]GTP single turnover assay with an IC 50 ∼ 4 μM and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into ml acetylcholine receptor-Gα q vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC 50 of RGS4 was increased from 1.5 to 7 nM. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys 20 to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activ. ity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.
| Original language | English |
|---|---|
| Pages (from-to) | 11115-11122 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 278 |
| Issue number | 13 |
| DOIs | |
| State | Published - Mar 28 2003 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Biochemistry
Cite this
Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid. / Ouyang, Ying Shi; Tu, Yaping; Barker, Sheryll A.; Yang, Fuyu.
In: Journal of Biological Chemistry, Vol. 278, No. 13, 28.03.2003, p. 11115-11122.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid
AU - Ouyang, Ying Shi
AU - Tu, Yaping
AU - Barker, Sheryll A.
AU - Yang, Fuyu
PY - 2003/3/28
Y1 - 2003/3/28
N2 - Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a Gα i1-[γ- 32P]GTP single turnover assay with an IC 50 ∼ 4 μM and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into ml acetylcholine receptor-Gα q vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC 50 of RGS4 was increased from 1.5 to 7 nM. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys 20 to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activ. ity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.
AB - Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a Gα i1-[γ- 32P]GTP single turnover assay with an IC 50 ∼ 4 μM and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into ml acetylcholine receptor-Gα q vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC 50 of RGS4 was increased from 1.5 to 7 nM. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys 20 to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activ. ity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.
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U2 - 10.1074/jbc.M212606200
DO - 10.1074/jbc.M212606200
M3 - Article
VL - 278
SP - 11115
EP - 11122
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 13
ER -