Abstract
Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for α subunits of the G i andor G q class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP 3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP 3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP 3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGSGα protein contact face, reduce binding affinity and inhibition by PIP 3. Calmodulin binds all RGS proteins tested in a Ca 2+-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca 2+calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP 3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP 3 act as allosteric inhibitors of RGS proteins, and Ca 2+calmodulin competition with PA and PIP 3 may provide an intracellular mechanism for feedback regulation of Ca 2+ signaling evoked by G-protein-coupled agonists.
| Original language | English |
|---|---|
| Pages (from-to) | 89-105 |
| Number of pages | 17 |
| Journal | Methods in Enzymology |
| Volume | 389 |
| DOIs | |
| State | Published - 2004 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
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Allosteric regulation of GAP activity by phospholipids in regulators of G-protein signaling. / Tu, Yaping; Wilkie, Thomas M.
In: Methods in Enzymology, Vol. 389, 2004, p. 89-105.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Allosteric regulation of GAP activity by phospholipids in regulators of G-protein signaling
AU - Tu, Yaping
AU - Wilkie, Thomas M.
PY - 2004
Y1 - 2004
N2 - Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for α subunits of the G i andor G q class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP 3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP 3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP 3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGSGα protein contact face, reduce binding affinity and inhibition by PIP 3. Calmodulin binds all RGS proteins tested in a Ca 2+-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca 2+calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP 3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP 3 act as allosteric inhibitors of RGS proteins, and Ca 2+calmodulin competition with PA and PIP 3 may provide an intracellular mechanism for feedback regulation of Ca 2+ signaling evoked by G-protein-coupled agonists.
AB - Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) for α subunits of the G i andor G q class of heterotrimeric G proteins. RGS GAP activity is inhibited by phosphatidic acid (PA), lysophosphatidic acid (LPA), and phosphatidylinositol 3,4,5-trisphosphate (PIP 3) but not by other phospholipids, phosphoinositides, or diacylglycerol. Both PA and PIP 3 can inhibit RGS4 GAP activity and their inhibition is additive, suggesting that PA and PIP 3 interact with different domains of RGS4. The N terminus of RGS4 (1-57 amino acids) is required for PA binding and inhibition. Mutation at Lys20, far from the RGS domain of RGS4, decreases PA-mediated inhibition of RGS4 by more than 85%. Amino acid substitutions in helix 5 within the RGS domain of RGS4, opposite to the RGSGα protein contact face, reduce binding affinity and inhibition by PIP 3. Calmodulin binds all RGS proteins tested in a Ca 2+-dependent manner at two sites, one in the N-terminal 33 amino acids and another in the RGS domain. Ca 2+calmodulin does not directly affect GAP activity of RGS4 but reverses PA and PIP 3-mediated inhibition. In summary, these results demonstrate that phospholipids such as PA and PIP 3 act as allosteric inhibitors of RGS proteins, and Ca 2+calmodulin competition with PA and PIP 3 may provide an intracellular mechanism for feedback regulation of Ca 2+ signaling evoked by G-protein-coupled agonists.
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UR - http://www.scopus.com/inward/citedby.url?scp=4344600505&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(04)89006-2
DO - 10.1016/S0076-6879(04)89006-2
M3 - Article
C2 - 15313561
AN - SCOPUS:4344600505
VL - 389
SP - 89
EP - 105
JO - Methods in Enzymology
JF - Methods in Enzymology
SN - 0076-6879
ER -