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J. Biol. Chem., Vol. 275, Issue 25, 18962-18968, June 23, 2000

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Ca²⁺/Calmodulin Reverses Phosphatidylinositol 3,4,5-Trisphosphate-dependent Inhibition of Regulators of G Protein-signaling GTPase-activating Protein Activity^*

Serguei G. Popov, U. Murali Krishna^§, J. R. Falck^§, and Thomas M. Wilkie^¶

From the  Pharmacology and ^§ Biochemistry Departments, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, February 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G_i and/or G_q class G protein  subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP₃) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP₃ but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP₃. Calmodulin binds all RGS proteins we tested in a Ca²⁺-dependent manner but does not directly affect GAP activity. Indeed, Ca²⁺/calmodulin binds a complex of RGS4 and a transition state analog of G_i1-GDP-AlF₄. Ca²⁺/calmodulin reverses PIP₃-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca²⁺/calmodulin competition with PIP₃ may provide an intracellular mechanism for feedback regulation of Ca²⁺ signaling evoked by G protein-coupled agonists.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Heterotrimeric G proteins link 7-transmembrane domain receptors to intracellular effector proteins in all mammalian cell types. These pathways are required for rapid responses to hormones and neurotransmitters. Signaling is initiated by agonist binding to receptors, which catalyze the exchange of GTP for GDP on the G subunit. Activated G-GTP and G subunits then regulate effector proteins that generate second messengers and subsequent downstream responses (reviewed in Ref. 1). The intensity, duration, and specificity of G protein-mediated signaling depend on a newly identified class of proteins, termed regulators of G protein signaling (RGS¹ proteins), which are GTPase-activating proteins (GAPs) for G subunits (Refs. 2-4 and reviewed in Ref. 5).

A diverse family of more than 20 RGS proteins are expressed in mammals (6). The RGS proteins share a common sequence feature of about 130 amino acids, referred to as the RGS domain (RGS box), which is responsible for GAP activity (7-9). Many RGS proteins, including RGS4, can accelerate GTP hydrolysis on both G_i and G_q class  subunits, whereas other RGS proteins are more selective catalysts (reviewed in Refs. 5, 10, and 11). Both full-length RGS4 and its RGS domain can inhibit Ca²⁺ signaling evoked by either G_i- or G_q-coupled agonists (12). The GAP activity of RGS proteins provided a molecular explanation of their role as inhibitors of G protein signaling, but mechanisms for how RGS GAP activity is regulated in cells remain obscure.

In several genetic systems, RGS proteins appear to act as feedback regulators of G protein signaling (7, 13, 14). Because RGS4 inhibits Ca²⁺ signaling in mammalian cells (11-17), we tested whether calmodulin, which regulates many Ca²⁺-responsive proteins stimulated by G_q-coupled agonists (18), could bind RGS4 and regulate its activity. We report that calmodulin binds in a Ca²⁺-dependent manner to all RGS proteins we tested, including RGS1, RGS2, RGS4, RGS10, RGS16, and GAIP. Surprisingly, Ca²⁺/calmodulin binding did not influence the GAP activity of RGS proteins.

Calmodulin regulates membrane association of many intracellular proteins (19-21), and it has been observed that RGS proteins associate with the inner surface of the plasma membrane (22-25). A cluster of positively charged residues on the surface of helixes 4 and 5 in the RGS domain was noted in the x-ray crystal structure of RGS4 (26) that could bind acidic lipids in the plasma membrane. Here we demonstrate that dipalmitoylphosphatidylinositol 3,4,5-trisphosphate (diC16-PIP₃ or PIP₃) binds RGS4 and inhibits its GAP activity in a concentration-dependent manner, in contrast to other phosphatidylinositol phosphates. PIP₃ inhibited GAP activity of other RGS proteins, including RGS1, RGS2, RGS10 and GAIP, but not RGS16. Amino acid substitutions in helix 5 of RGS4 reduced PIP₃ binding (10-fold) and PIP₃-dependent inhibition of GAP activity. A potential mechanism for feedback regulation of G protein-mediated Ca²⁺ signaling was suggested by the observation that PIP₃-dependent inhibition of GAP activity was reversed by Ca²⁺/calmodulin. The concerted action of Ca²⁺/calmodulin and PIP₃ may regulate RGS GAP activity to initiate [Ca²⁺]_i oscillations evoked by G protein-coupled agonists.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Calmodulin, Peptides, and Inositol Lipids-- Bovine brain calmodulin and calmodulin covalently attached to agarose beads (CaM-agarose) were from Sigma. Peptide P_1-33, MCKGLAGLPASCLRSAKDMKHRLGFLLQKSDSC, was described (12), and a scrambled sequence of P_1-33, MLDQAPLKSKSACAGKLRMHGCLSFGLRLKCSD, P_1-33, was synthesized by C. Slaughter (UT Southwestern). L--Phosphatidylcholine (PC), L--phosphatidylserine (PS), L--phosphatidyl-sn-glycerol from egg yolk lecithin (PG), L--phosphatidylinositol 4,5-bisphosphate (PIP₂), L--phosphatidylinositol 4-phosphate (PIP), L--phosphatidylinositol (PI), all from bovine brain, D-myo-inositol 1,3,4,5-tetrakisphosphate (IP₄), and 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG) were purchased from Sigma. L--Phosphatidylethanolamine (PE) from bovine liver was purchased from Avanti Polar Lipids. Dihexadecanoylphosphatidylinositol 3,4,5-trisphosphate (diC16-PIP₃), diC16-3,5-AP₂ dioctanoylphosphatidylinositol 3,4,5-trisphosphate (diC8-PIP₃), dioctanoylphosphatidylinositol 3,4-bisphosphate (diC8-3,4-PIP₂), dioctanoylphosphatidylinositol 3,5-bisphosphate (diC8-3,5-PIP₂), and dioctanoylphosphatidylinositol 4,5-bisphosphate (diC8-4,5-PIP₂) were synthesized as described (27-29).

Production and Purification of Recombinant Proteins-- G_i1, RGS4, RGS10, GAIP, RGS4, and RGS4 mutants were expressed as His₆-tagged proteins and purified using a Ni²⁺-nitrilotriacetic acid-agarose (9). RGS16 expression and purification were similar to RGS4 (9). Recombinant RGS1 and RGS2 were kindly provided by Drs. K. J. Blumer, S. Heximer (Washington University), and D. Forsdyke (Queens University), respectively (16). The fragment coding for mutant RGS4 K112E/K113E was generated by polymerase chain reaction using the oligonucleotide containing the above mutation as a primer and cloned into the pQE60 expression vector (Qiagen) as described for RGS4 (9). RGS4 C95V protein was kindly provided by Dr. E. Ross (UT Southwestern).

Ca²⁺/Calmodulin Binding-- Concentration of calmodulin was measured spectrophotometrically using the extinction coefficient 3060 M¹ cm¹ at 278 nm in presence of 1 mM EGTA (30). Concentration of RGS4 was measured using Bradford reagent from Bio-Rad. Band shift gel analysis of calmodulin binding to RGS4 and RGS2 in 4 M urea, in the presence of 0.1 mM Ca²⁺ or 2 mM EDTA, was carried out as described (30). RGS binding to calmodulin-agarose was detected by SDS-PAGE of supernatants as follows: 15 µl of wet calmodulin-agarose beads (26 µg of CaM) washed with buffer (10 mM HEPES, pH 7.4, 0.1 mM CaCl₂, 1 mM DTT) were pelleted; the supernatant was removed, and the beads were mixed with RGS4 (1.5 nmol) equal in amount to coupled calmodulin in a final volume 30 µl of buffer. The suspension was incubated at room temperature with constant agitation. After 30 min, the beads were washed twice with 500 µl of buffer. The suspension was brought to the original volume and incubated for 10 min. 500 mM EGTA and 5 M NaCl were added to final concentrations 2 and 50 mM, respectively, and the suspension was incubated for additional 10 min. Finally, 5 µl of the SDS-PAGE loading buffer was added to the beads and incubated another 10 min. Equal volume aliquots of supernatant were withdrawn after each incubation step, followed by separation by SDS-PAGE and Coomassie Blue staining.

Fluorescent detection of RGS4-calmodulin interaction was performed using Perkin-Elmer LS 50B and Hitachi F-2000 fluorescent spectrophotometers at 25 ± 0.1 °C. Both excitation and emission slit widths were 10 nm. For internal RGS4 tryptophan fluorescence measurements, the excitation and the emission wavelengths were 283 and 336 nm, respectively. Typically, aliquots of CaM solution were gradually added to the solution of RGS4 in the assay cuvette with constant agitation, and after each addition the mixture was equilibrated inside the instrument with the excitation beam shutter closed. The fluorescence measurements were made after 5-15 min. The excitation beam shutter was opened only long enough to get accurate readings. Measurements were repeated until reproducible readings were obtained. Data in Fig. 2 were corrected for dilution (which did not exceed 7%) and for fluorescence of calmodulin alone.

Calmodulin was dansylated as described (31) and extensively dialyzed against 10 mM HEPES, pH 7.4. Fluorescence measurements of dansylated calmodulin (dansyl-CaM) were at 335 (excitation) and 500 nm (emission). All measurements were corrected for background fluorescence observed in control experiments. The Scatchard plot analysis of the fluorescence affinity measurement data indicated a single binding site interaction (or 2 sites with similar affinities) and fit well to linear approximations within the experimental error.

RGS GAP Assays-- GAP assays were carried out in a soluble single turnover system with G_i1 as described (2, 9) with minor modifications. Briefly, 250-500 nM G_i1 in the assay buffer (10 mM HEPES, pH 8.0, 5 mM EDTA, 2 mM DTT) was loaded with [-³²P]GTP at 30 °C for 20 min. The solution was placed on ice for 5 min, and all subsequent reactions were carried out on ice in a cold room. A drop of RGS protein (5-10 µl) was placed onto a wall of reaction tube next to a 5-µl drop of solution, 500 mM MgCl₂, 5 mM cold GTP, and the GAP reaction was started by addition of 175 µl of G_i1-GTP solution. The final concentration of free Mg²⁺ ions in the reaction mix was 3-4 mM. To study the influence of Ca²⁺/calmodulin on RGS4 GAP activity with G_i1-GTP, RGS4 was preincubated with calmodulin in 10 mM HEPES, pH 8.0, 1 mM CaCl₂, 2 mM DTT on ice for 20 min. Then 175 µl of G_i1 loaded with [-³²P]GTP was added with 5 µl of 500 mM GTP, 475 mM MgCl₂, 25 mM CaCl₂.

Lipid Vesicles-- To prepare small unilamellar lipid vesicles (SUVs), chloroform-soluble lipids were evaporated under a stream of nitrogen into a dry film, and resuspended by vortexing in a sonication buffer, 10 mM HEPES, pH 8.0, 0.1 mM EDTA, 2 mM DTT, essentially as described (32, 33). SUVs were generated by sonication of a lipid suspension (2 mg/ml total lipid) in a water bath at room temperature for 10-15 min. To prepare SUVs containing chloroform-insoluble diC16-PIP₃, the suspension of diC16-PIP₃ in the sonication buffer (10 mg/ml) was added to the dry film of other lipids, vortexed vigorously for 1 min, and sonicated after addition of the appropriate amount of the sonication buffer. Aggregated material was removed from the preparations of SUVs by centrifugation at 10,000 × g for 10 min. The micelles of diC16-PIP₃ were obtained by its brief sonication in a buffer solution (32).

Surface Plasmon Resonance-- Surface plasmon resonance measurements were performed using BIAcore 1000 instrument (BIAcore, Inc.) at 25 °C. RGS protein was immobilized on the surface of the carboxymethylated dextran chip (CA-5) using standard carbodiimide chemistry in accordance with manufacturer's instructions. Lipid dissolved in the running buffer, 10 mM HEPES, pH 8.0, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant NP20, was injected over the chip surface with a flow rate 5 µl/min. At least three different concentrations were used. Control injections were made with a blank chip without coupled RGS. The amount of coupled protein was 2500 (RGS4), 1580 (RGS4 K112E/K113E), and 2900 response units (RU) (RGS16). The regeneration of the chip after each binding experiment was achieved by injecting 0.01% SDS in running buffer. The data were analyzed using the BIAevaluation 2.1 software (BIAcore, Inc.). The binding curves showed a moderate heterogeneity of both association and dissociation phases. The minor components identified in the analysis of association and dissociation (less than 20% of total signal) were ignored. A relatively fast transition process evident at the beginning of the dissociation phase was not studied. The dissociation constant, K_d, was calculated for each sensorgram using kinetic constants k_a and k_d. The average values are shown in Table IV.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Ca²⁺/Calmodulin Binds RGS Proteins-- To investigate the role of Ca²⁺ in feedback regulation of G protein signaling by RGS proteins, we characterized two potential calmodulin binding regions in RGS4 as follows: one in the N-terminal 33 amino acids and another between residues 99-113, in helixes 4 and 5 of the RGS domain (4Box). These regions contain amphiphatic sequences with bulky hydrophobic residues at certain positions and clusters of positively charged amino acids similar to calmodulin-binding sites in other proteins (Table I; see Ref. 18). We found that RGS4 bound calmodulin in a Ca²⁺-dependent manner (the complex was dissociated by 2 mM EGTA) in a standard band shift assay for calmodulin binding (Fig. 1; Ref. 30). RGS4 binding to Ca²⁺/calmodulin was corroborated using calmodulin coupled to agarose beads. RGS4 binding to calmodulin-agarose beads required Ca²⁺ and was stable to buffer washes, but bound RGS4 could be eluted from beads either with EGTA or SDS (Fig. 1C). We found that a previously characterized amphipathic calmodulin-binding peptide from CaM kinase II (34) competed with RGS4 binding to calmodulin-agarose beads (Fig. 1E, 4th and 5th lanes). An RGS4 N-terminal 33 amino acid peptide (P_1-33), which conveys high affinity and receptor-selective regulation of G_q signaling (12), bound Ca²⁺/calmodulin (Fig. 1D) and competed with RGS4 for binding to calmodulin-agarose beads (Fig. 1E, 6th and 7th lanes). By contrast, a scrambled sequence composed of the same amino acids (P_1-33) did not bind to calmodulin beads (data not shown) and did not compete with RGS4 binding (Fig. 1E, 8th and 9th lanes). Interestingly, RGS4 apparently formed a heterotrimeric complex with G_i1-GDP-AlF₄ and Ca²⁺/calmodulin (Fig. 1F), consistent with their predicted distinct binding sites on RGS4.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   RGS4 binding to CaM is Ca2+-dependent. Band shift analyses (in 4 M urea) shows that increasing the molar ratio of RGS4 recruits CaM into a complex of RGS4-Ca2+/CaM (A). EGTA (2 mM) dissociates the complex (B). RGS4 and P1-33 binding to CaM-agarose beads is dissociated by EGTA (C and D). Peptides CaM kinase II and P1-33 but not P1-33 compete with RGS4 for Ca2+/CaM binding. Pairs of lanes for each peptide show the relative yield of RGS4 from supernatants after incubation with beads (s) and after SDS elution from beads (b) (E). Binding of the RGS4-Gi1-GDP-AlF4 complex to Ca2+/CaM-agarose beads (F). Proteins were visualized on Coomassie-stained PAGE gels.

We used fluorescence spectroscopy to quantitate binding interactions between RGS4 and Ca²⁺/calmodulin. The fluorescence of two tryptophan residues within the RGS domain of RGS4 (4Box) was quenched by titration with Ca²⁺/calmodulin (which lacks tryptophan), indicative of RGS4-Ca²⁺/calmodulin binding in solution (Fig. 2). A sharp inflection in the titration curve indicates formation of a stable, equimolar complex between Ca²⁺/calmodulin and 4Box (20 mM NaCl, 10 mM HEPES, pH 7.4). Upon further addition of Ca²⁺/calmodulin to 4Box, the slope of the fluorescence titration curve paralleled that of unbound RGS4 and Ca²⁺/calmodulin (calculated as the sum of their fluorescence signals measured separately). This behavior is consistent with the prediction of a single Ca²⁺/calmodulin-binding site in the RGS domain. By contrast, in the absence of salt, the fluorescence intensity of RGS4 (Fig. 2) or 4Box (data not shown) did not change at higher molar ratios of Ca²⁺/calmodulin, suggesting additional, low affinity calmodulin-binding sites on RGS4. These low affinity interactions are probably electrostatic because they were not detected at higher ionic strength (Fig. 2, 4Box, and data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Ca2+/calmodulin-binding site in the RGS domain. Relative fluorescence intensity of mixtures of CaM with RGS4 or the RGS4 GAP domain (4Box) recorded after incubation in 10 mM HEPES, pH 7.4, 0.1 mM CaCl2, 1 mM DTT with 20 mM NaCl (4Box) or no added salt (RGS4). The inflection point at the 1:1 molar ratio indicates a single high affinity binding site. RGS4 exhibits additional low affinity binding of Ca2+/CaM in low salt. Filled squares indicate the sum of fluorescent intensities of CaM and RGS4 assayed separately.

In addition to the RGS domain, Ca²⁺/calmodulin apparently binds to the N-terminal 33 amino acids of RGS4 because the peptide P_1-33 binds in a Ca²⁺-dependent manner both to calmodulin beads (Fig. 1) and in solution, as detected by two-dimensional heteronuclear single quantum coherence NMR using [¹⁵N]calmodulin.² Although P_1-33 has no residues with fluorescent properties convenient for affinity measurements, we used a dansylated derivative of calmodulin (dansyl-CaM, see Ref. 31) which allowed us to compare the binding properties of P_1-33 and RGS4 in similar conditions. The apparent K_d values (Table II) were calculated from Scatchard linear transformations of titration curves (Fig. 3). In the presence of 100 mM NaCl, RGS4 and P_1-33 bind to Ca²⁺/dansyl-CaM with similar affinities (K_d 5 µM). The affinity of P_1-33 binding within the accuracy of measurements did not change with ionic strength, whereas RGS4 bound Ca²⁺/dansyl-CaM almost 5 times stronger than P_1-33 in 20 mM NaCl, consistent with our observations that a decrease in salt concentration strengthened the interaction of RGS4 with Ca²⁺/calmodulin beads.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dansylated Ca2+/calmodulin binds RGS4 N terminus (P21-33). Fluorescent titration of 0.5 µM dansylated CaM (dansyl-CaM) with P1-33 peptide (A) or RGS4 (B) in 10 mM HEPES, pH 8.0, 1 mM DTT, 0.1 mM CaCl2 at different concentrations of NaCl as follows: 20 mM (open circles), 50 mM (filled circles), and 100 mM (open triangles). EGTA (5 mM, squares) reduced dansyl-CaM binding in 20 mM NaCl. Fraction of bound dansyl-CAM was calculated based on fractional decrease in fluorescence intensity.

Calmodulin binds to many RGS proteins in a Ca²⁺-dependent manner but with different salt dependences. Binding assays indicated that RGS4, RGS16, and GAIP had similar affinities toward calmodulin-agarose beads in 20 mM NaCl, whereas RGS10 interaction with Ca²⁺/calmodulin was relatively weak (even without salt). Only RGS1 and RGS2 bound to calmodulin-agarose beads in high salt (150 mM KCl). Stable interaction was corroborated by band shift analysis that revealed an RGS2-Ca²⁺/calmodulin complex in high ionic strength running buffer with 4 M urea, 275 mM Tris-HCl, pH 8.3 (data not shown).

Calmodulin Does Not Influence RGS4 GAP Activity-- To test the biological relevance of Ca²⁺/calmodulin binding to RGS proteins, we studied its effect on RGS4 and RGS1 GAP activity. We found that neither 1 mM Ca²⁺ alone nor 1.6 µM Ca²⁺/calmodulin preincubated with either RGS protein altered their GAP activity toward G_i1 in a single turnover assay (Fig. 4 and data not shown). Because the N terminus of RGS4 is not required for GAP activity (9) but binds calmodulin (Fig. 1, D and E), we also tested GAP activity of an N-terminal deletion mutant that lacked the first 57 residues of RGS4 but retained the calmodulin-binding site within the RGS domain (R4N; Fig. 4B). The GAP activities of full-length RSG4 and R4N were unaffected by Ca²⁺/calmodulin in the single turnover assay (Fig. 4). These results indicated that Ca²⁺/calmodulin binding neither sterically blocked G_i1 binding nor irreversibly changed the conformation of the RGS4-G_i1 interface. Considering the stability of RGS4 binding to Ca²⁺/calmodulin beads (Fig. 1), it seems improbable that GAP activity resulted from the rapid and transient displacement Ca²⁺/calmodulin from RGS4 by G_i-GTP. Indeed, we found that G_i1-GDP-AlF₄, which mimics a transition state of RGS4-catalyzed G-GTP hydrolysis (K_d = 0.6 nM at 25 °C, Ref. 9), binds as a complex with RGS4 and Ca²⁺/calmodulin-agarose beads (Fig. 1E). This complex was stable to extensive washing with buffer, but RGS4-G_i1-GDP-AlF₄ was eluted from Ca²⁺/calmodulin beads by 1% SDS. G_i1-GDP-AlF₄ did not bind to Ca²⁺/calmodulin in the absence of RGS4. These results indicate that a heterodimeric complex of Ca²⁺/calmodulin-RGS4 retains GAP activity. We therefore favor a model in which calmodulin binds surface residues of the RGS domain without substantially altering the RGS4 conformation.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   RGS4 GAP activity is not inhibited by Ca2+/calmodulin. RGS4 (0.8 µM in A) or R4N (N-terminal 56-amino acid truncation; 20 µM in B) was preincubated with Ca2+/CaM (1.6 µM in A or 75 µM in B) before initiating a single turnover GAP assay. Open circles, RGS4 with CaM; closed squares, RGS4 without CaM; closed circles, basal Gi1-GTP hydrolysis. Final RGS4 concentrations were 22 nM (A) and 220 nM (B).

PIP₃ Inhibits RGS GAP Activity-- Previous studies indicated that brief dialysis of recombinant RGS4 into patch clamped pancreatic acinar cells potently inhibited Ca²⁺ signaling evoked by G_i- and G_q-coupled receptor agonists (12, 16). This suggested the possibility that endogenous RGS proteins might be relatively inactive prior to agonist stimulation of Ca²⁺ signaling and that recombinant RGS proteins escaped this inhibition. To identify inhibitors of RGS4 GAP activity on G_i1 in the single turnover assay, we tested various compounds related to and including either the substrate or products of PLC. We found that RGS4 GAP activity was inhibited by preincubation with an analog of PIP₃, diC16-PIP₃ (30 µM PIP₃, at 0 °C for several minutes, Fig. 5A), or phosphocholine vesicles containing 20% PIP₃ (Fig. 5B). Inhibition of RGS4 GAP activity was dependent on the concentration of PIP₃ (Fig. 5C). The kinetic curve of GTP hydrolysis in the presence of PIP₃ closely approximated the basal activity of G without RGS4. This behavior indicated negligible dissociation of PIP₃ from RGS4 during the assay (5 min) because GTP hydrolysis was initiated by a rapid 20-fold dilution of the RGS-PIP₃ incubation mix into a solution containing G_i1-GTP. In control experiments, the intrinsic GTPase activity of G_i1 was unaffected by PIP₃ (Fig. 5D). The GAP activity of each RGS protein that was tested in the single turnover assay, except RGS16, was inhibited by PIP₃ (Fig. 5D and data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   DiC16-PIP3 inhibits RGS4 GAP activity. Single turnover assay with Gi1-GTP. RGS4 (2 µM) was preincubated with 30 µM diC16-PIP3 micelles (open squares) or 70 µM diC16-PIP3 (open circles) on ice in 6-µl volume for 20 min. Control reactions contained no PIP3 (squares) or no RGS4 (filled circles) (A). Single turnover assay with RGS4 and Gi1-GTP in the presence of PC lipid vesicles contained 20% diC16-PIP3 (open circles) or 20% PIP2 (filled squares). RGS4 (0.72 µM) was preincubated with 10-µl vesicles on ice in 11-µl volume for 20 min. Control reactions contained no lipid (open squares) or neither RGS nor lipid (filled circles) (B). DiC16-PIP3 inhibition of RGS4 GAP activity is concentration-dependent in GAP assays with PIP3 (0.9, 2.7, and 9.1%) incorporated into PC/PG (9:1) vesicles (C). GAP activity of RGS proteins in the presence of diC16-PIP3. RGS1 (2 µM), RGS10 (0.5 µM), GAIP (0.24 µM), and RGS16 (2 µM) were preincubated with or without 200 µM PIP3 on ice for 20 min. GAP activity was assayed as in A.

By contrast to PIP₃ inhibition of RGS GAP activity, no effect was observed following coincubation of RGS4 with 400 µM PIP₂ from bovine brain (Fig. 5B), and only 2-3-fold inhibition was observed following coincubation with PIP₂ micelles (9 mM). We barely detected the inhibitory activity of 4-mono phosphorylated phosphatidylinositol phosphate, PIP (9 mM). As summarized in Table III, several synthetic PIP₂ lipids were also without effect (assayed at 400 µM in phosphocholine vesicles), including dioctanoylphosphatidylinositol 3,4-bisphosphate (diC8-3,4-PIP₂), dioctanoylphosphatidylinositol 3,5-bisphosphate (diC8-3,5-PIP₂), and dioctanoylphosphatidylinositol 4,5-bisphosphate (diC8-4,5-PIP₂). RGS4 GAP activity was also not affected by a lipid head group derivative, 1-stearoyl-2-arachidonoyl-sn-glycerol (diacylglycerol, DAG), which is one of the reaction products of PLC. No inhibitory activity was detected using PI or highly charged head group derivatives of PIP₃ lacking the fatty acyl moieties, including inositol 1,3,4,5-tetrakisphosphate (IP₄) and glycerophosphoinositol 3,4,5-trisphosphate (IP₃, the other reaction product of PLC; data not shown). PIP₃ binding to Rac1 and RhoA was shown to have similar requirements for both electrostatic and hydrophobic interactions (35). Surprisingly, we found that dioctanoylphosphatidylinositol 3,4,5-trisphosphate (diC8-PIP₃), which only differed from PIP₃ (diC16-PIP₃) in the length of the fatty acid chains, did not inhibit RGS4 GAP activity; nor did diC16-3,5-PIP₂. Thus, diC16-PIP₃ is the only phospholipid which inhibited RGS4 GAP activity, and its activity appears to require both the long chain fatty acid moiety and the highly charged head group.

RGS4 Is a PIP₃-binding Protein-- Protein-lipid binding affinities were estimated by surface plasmon resonance measurements on the BIAcore (Biacore, Inc). RGS4 coupled to the carboxymethylated dextran surface of the BIAcore chip bound diC16-PIP₃ (Fig. 6A) but not diC8-PIP₃, PIP₂, or other phosphoinositides, consistent with the observation that RGS GAP activity was most sensitive to inhibition by diC16-PIP₃. The association phase of diC16-PIP₃ binding was typically complete within several minutes, whereas dissociation was slow (Fig. 6A). No diC16-PIP₃ binding was detected on a blank chip. The K_d value of diC16-PIP₃ binding to RGS4 (44 ± 19 nM; Table IV) was calculated from the on and off rates extracted from the binding curves (Fig. 6A, and data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   RGS4 binds PIP3. BIAcore sensorgrams of diC16-PIP3 (4 µM) binding to RGS proteins immobilized on the chip (background binding subtracted). Binding curves for RGS16 and RGS4 K112E/K113E were normalized to 2500 RU of coupled RGS4 (A). The GAP activities of mutant RGS4 K112E/K113E (B, open bars) and RGS16 after 90 s (see Fig. 5D) are less sensitive than wild type RGS4 (B, solid bars) to inhibition by PC/PIP3 vesicles (8:2). Single turnover GAP assay as in Fig. 5.

Ca²⁺/CaM Antagonizes the PIP₃ Inhibition of RGS4 GAP Activity-- The positively charged patch on the surface of helixes 4 and 5 in the RGS domain of RGS4 (residues 99-113; Ref. 26) appeared to be a good candidate for binding not only to calmodulin but also to PIP₃. We found that PIP₃ inhibition of RGS4 GAP activity was reversed by coincubation of RGS4 and PIP₃ (micelles or phosphocholine vesicles) with Ca²⁺/calmodulin (Fig. 7). Ca²⁺/calmodulin and PIP₃ apparently compete for binding to helixes 4 and 5 in RGS4. To test this model further, we introduced amino acid substitutions of glutamate for lysine residues at positions 112 and 113 in helix 5 of RGS4. This mutant protein (RGS4 K112E/K113E) retained GAP activity and calmodulin binding, but it bound PIP₃ with almost 10-fold lower affinity than did wild type RGS4 (Table IV). Similarly, low binding affinity was observed with RGS16. The comparatively weak binding of these proteins to PIP₃ correlated with their relative insensitivity to PIP₃ inhibition of GAP activity (Figs. 5D and 6B). Helixes 4 and 5 are conserved in many RGS domains (Table I) and may provide an important regulatory feature of RGS proteins because Ca²⁺/calmodulin has been shown to serve as a molecular switch that regulates lipid-protein interactions (19-21).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Ca2+/CaM reverses inhibition of RGS4 GAP activity by diC16-PIP3. Single turnover GAP assay with PIP3 incorporated into lipid vesicles, as in Fig. 5C. Before initiating the assay, RGS4 was incubated for 20 min on ice with PC/PIP3 vesicles (8:2) alone or with of 100 µM Ca2+/CaM (A). Single turnover GAP assay with PIP3 micelles. Before initiating the assay, RGS4 (5 µM) was incubated for 20 min on ice with 40 µM PIP3 (B and C, open circles), 40 µM PIP3, and 100 µM Ca2+/CaM (B, open squares), or 40 µM PIP3, 100 µM CaM, and 2 mM EGTA (C, open squares). Control incubations contained no lipid (B and C, filled squares) or no RGS4 (B and C, filled circles). 2 µl of each incubation mix were used for GAP assay.

PIP₃ Inhibits GAP Activity of Palmitoylation-resistant RGS Proteins-- We propose that PIP₃-mediated inhibition of GAP activity acts by a concerted mechanism in which the highly charged head group interacts with the RGS domain residues in helixes 4 and/or 5 to position the palmitoyl moiety of PIP₃ near its binding site. Palmitoylation of a nearby cysteine residue inhibited RGS4 and RGS10 GAP activity (36). This cysteine residue in helix 4 (Cys⁹⁵ in RGS4) is conserved in the RGS domain of all mammalian RGS proteins except RGS6 and RGS7 (6). Substitution of cysteine for valine (C95V) prevents covalent modification by palmitate at this position in RGS4. GAP activity of the C95V mutant protein is equivalent to wild type protein, but it is not inhibited by palmitoylation (36). By contrast, the GAP activity of RGS4 C95V is as sensitive to PIP₃-mediated inhibition as is wild type protein (Fig. 8A). The GAP activities of RGS16 and the mutant RGS4 K112E/K113E, which were relatively insensitive to inhibition by PIP₃, responded like wild type protein to inhibition by palmitoylation (Fig. 8, B and C). In contrast to RGS4 interaction with PIP₃, inhibition of GAP activity by palmitoylation was not prevented by addition of Ca²⁺/calmodulin (Fig. 8D). This is presumably because Ca²⁺/calmodulin competes with PIP₃ binding to RGS4 but cannot displace the covalent modification of RGS4 by palmitate. The feedback mechanisms that regulate the palmitoylation of RGS proteins in vivo are unknown, but we propose that PIP₃-mediated inhibition of RGS GAP activity may be reversed in a Ca²⁺-dependent manner through binding of Ca²⁺/calmodulin.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   DiC16-PIP3 inhibits GAP activity of a palmitoylation-resistant RGS4 mutant. RGS4 C95V (1 µM) was preincubated with 80 µM PIP3 (open circles) on ice in 5-µl volume for 20 min. Control reaction contained no PIP3 (squares) or no RGS (filled circles) (A). RGS4 K112E/K113E GAP activity is inhibited by palmitoylation. RGS4 K112E/K113E (3 µM) was incubated with 50 µM palmitoyl-CoA (Pal-CoA) for 1 h at 30 °C (open circles). 2 µl of the reaction mix were used in GAP assay. Control reactions contained no RGS4 K112E/K113E (filled circles) or no palmitoyl-CoA (filled squares) (B). RGS16 is susceptible to inhibition by palmitoylation. RGS16 (6 µM) was incubated with 50 µM palmitoyl-CoA for 1 h at 30 °C (open circles). 2 µl of the reaction mix were used in GAP assay. Control reactions contained no RGS16 (filled circles) or no palmitoyl-CoA (filled squares) (C). Ca2+/CaM does not reverse inhibition of RGS by palmitoylation. RGS4 (2 µM) was preincubated with 25 µM palmitoyl-CoA for 1 h at 30 °C alone (open circles) or in the presence of 100 µM Ca2+/CaM (triangles). 3 µl of each reaction mix were used in GAP assay. Control reactions contained no RGS4 (filled circles) or no palmitoyl-CoA (Pal-CoA) (filled squares) (D). Single turnover assay as in Fig. 5A.

Feedback Regulation of RGS GAP Activity by Ca²⁺/Calmodulin and PIP₃-- Negative regulation of RGS GAP activity by PIP₃ may be part of a reset mechanism that allows a new wave of G protein signaling in response to agonist. G protein-coupled receptors, such as formyl-methionyl-leucyl-phenylalanyl receptors in neutrophils, can elicit both Ca²⁺-release and a rapid and large accumulation of PIP₃ by activating the effector proteins PLC and PI-3 kinase, respectively (reviewed in Ref. 37). Ca²⁺ release from internal stores is one of the initial responses to PLC activation either by G_q or G from G_i class G proteins. As the local concentration of Ca²⁺ elevates in response to PLC activity, we postulate that Ca²⁺/calmodulin binding to RGS4 and other RGS proteins displaces PIP₃ to restore GAP activity (modeled in Fig. 9). Ca²⁺/calmodulin binding may also enhance GAP activity by relocating RGS proteins within the receptor signaling complex to be in proximity to their G-GTP substrates. Feedback inhibition of G protein-mediated PLC activation by RGS proteins would allow [Ca²⁺]_i to decrease and promote the dissociation of calmodulin from RGS proteins. We hypothesize that PIP₃ released after receptor stimulation could then bind RGS proteins to inhibit their GAP activity. If agonist stimulation persists, this may reactivate G protein signaling and allow another burst of Ca²⁺ release from internal stores. If agonist is no longer present, PIP₃-mediated inhibition of RGS GAP activity may reset the signaling pathway to allow a robust cellular response to subsequent agonist stimulation. Feedback regulation of RGS GAP activity may provide an intracellular mechanism to initiate oscillations over a wide range of frequencies.

View larger version (31K): [in this window] [in a new window]   Fig. 9.   A model of RGS regulation by Ca2+calmodulin and PIP3. Agonist-bound receptor promotes GTP binding to the G subunit and subsequent dissociation of G and G. The effector protein PLC may be activated by either Gq or G from Gi class proteins. PLC catalyzes the hydrolysis of PIP2 to produce DAG and IP3, which binds IP3R to release Ca2+ from intracellular stores. We propose that endogenous RGS proteins may be inactive prior to agonist-evoked Ca2+ signaling, but as the local concentration of intracellular Ca2+ elevates, it binds calmodulin, which can displace PIP3 from helixes 4 and 5 of RGS proteins, and thereby restores RGS GAP activity. Calmodulin binding may also reposition RGS within the receptor complex to enhance activity. RGS-mediated inhibition of G protein signaling would decrease [Ca2+]i allowing dissociation of calmodulin and rebinding of PIP3 to inhibit RGS GAP activity.

	ACKNOWLEDGEMENTS

We thank Y. Tu for RGS4C95V protein; K. Chapman for [-³²P]GTP; J. Rizo-Rey, K. Luby-Phelps, I. Fernandez, and C. Wigley for their contributions to NMR analysis of the Ca²⁺/calmodulin-RGS4 complex; C. Slaughter for peptide synthesis; and E. Ross, S. Muallem, and colleagues for discussions and comments on the manuscript.

	FOOTNOTES

^* This work was supported in part by grants from the R. A. Welch Foundation and National Institutes of Health Grants GM31278 (to J. R. F.) and DK47890 (to T. M. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of the American Heart Association Established Investigator award. To whom correspondence should be addressed: Pharmacology Dept., University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-8581; E-mail: thomas.wilkie@email.swmed.edu.

Published, JBC Papers in Press, March 28, 2000, DOI 10.1074/jbc.M001128200

² K. L.-P. I. Fernandez, S. G. Popov, and J. Rizo-Rey, unpublished data.

	ABBREVIATIONS

The abbreviations used are: RGS, regulators of G protein signaling; PIP₃ or diC16-PIP₃, dihexadecanoylphosphatidylinositol 3,4,5-trisphosphate; PIP₂, L--phosphatidylinositol 4,5-bisphosphate; PIP, L--phosphatidylinositol 4-phosphate; PI, L--phosphatidylinositol; DAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; Gro-IP₃, glycerophosphoinositol 3,4,5-trisphosphate; diC8-PIP₃, dioctanoylphosphatidylinositol 3,4,5-trisphosphate; diC8-3, 4-PIP₂, dioctanoylphosphatidylinositol 3,4-bisphosphate; diC8-3, 5-PIP₂, dioctanoylphosphatidylinositol 3,5-bisphosphate; diC8-4, 5-PIP₂, dioctanoylphosphatidylinositol 4,5-bisphosphate; PC, L--phosphatidylcholine; PS, L--phosphatidyl-L-serine; PG, L--phosphatidyl-sn-glycerol; CaM, calmodulin; GAP, GTPase-activating protein; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; dansyl-CaM, dansylated calmodulin; PAGE, polyacrylamide gel electrophoresis; SUVs, small unilamellar lipid vesicles; PLC, phospholipase C; RU, response units.

	REFERENCES

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