Gelsolin Binding to Phosphatidylinositol 4,5-Bisphosphate Is Modulated by Calcium and pH*

(Received for publication, April 24, 1997, and in revised form, May 22, 1997)

Keng-Mean Lin Dagger , Elizabeth Wenegieme Dagger , Pei-Jung Lu §, Ching-Shih Chen § and Helen L. Yin Dagger

From the Dagger  Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas and the § Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The actin cytoskeleton of nonmuscle cells undergoes extensive remodeling during agonist stimulation. Lamellipodial extension is initiated by uncapping of actin nuclei at the cortical cytoplasm to allow filament elongation. Many actin filament capping proteins are regulated by phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed by phospholipase C. It is hypothesized that PIP2 dissociates capping proteins from filament ends to promote actin assembly. However, since actin polymerization often occurs at a time when PIP2 concentration is decreased rather than increased, capping protein interactions with PIP2 may not be regulated solely by the bulk PIP2 concentration. We present evidence that PIP2 binding to the gelsolin family of capping proteins is enhanced by Ca2+. Binding was examined by equilibrium and nonequilibrium gel filtration and by monitoring intrinsic tryptophan fluorescence. Gelsolin and CapG affinity for PIP2 were increased 8- and 4-fold, respectively, by µM Ca2+, and the Ca2+ requirement was reduced by lowering the pH from 7.5 to 7.0. Studies with the NH2- and COOH-terminal halves of gelsolin showed that PIP2 binding occurred primarily at the NH2-terminal half, and Ca2+ exposed its PIP2 binding sites through a change in the COOH-terminal half. Mild acidification promotes PIP2 binding by directly affecting the NH2-terminal sites. Our findings can explain increased PIP2-induced uncapping even as the PIP2 concentration drops during cell activation. The change in gelsolin family PIP2 binding affinity during cell activation can impact divergent PIP2-dependent processes by altering PIP2 availability. Cross-talk between these proteins provides a multilayered mechanism for positive and negative modulation of signal transduction from the plasma membrane to the cytoskeleton.


INTRODUCTION

Phosphoinositides are important in signal transduction, both as precursors to signaling molecules and as physical anchors and regulators of proteins (1, 2). Among these, the D4 phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2),1 has been implicated as a potential mediator of actin cytoskeletal rearrangements (3, 4). PIP2 modulates many actin regulatory proteins. These include the following: actin severing and/or capping proteins (gelsolin (5), CapG (6), and capping protein (also known as Cap Z) (7)), monomer-binding proteins (profilin (8) and cofilin (9)), and other actin-binding proteins (alpha -actinin (10) and vinculin (11)). It has been hypothesized that PIP2 induces explosive actin assembly by dissociating capping proteins from filament ends and releasing actin monomers from actin-sequestering proteins (3, 7, 12). The involvement of PIP2 in actin polymerization is supported by recent experiments that show that Rac1 and RhoA, monomeric GTPases of the Rho family that have well defined effects on the cytoskeleton (13), stimulate the synthesis of PIP2 (14-16). Furthermore, manipulations that alter the availability of PIP2 in cells have profound effects on agonist and/or Rac1-induced filament end capping, actin polymerization, and cell motility (16, 17). However, although the time courses of PIP2 hydrolysis and recovery correlate in some cells (16, 18), they do not in most of the cells examined (19-21). Particularly puzzling is the finding that, in many cells, actin polymerizes at a time when PIP2 level is reduced, rather than increased, as would be expected if uncapping and monomer desequestration are initiated by PIP2. To explain this discrepancy, it is often hypothesized that local PIP2 availability can be enhanced by compartmentalization or differential turnover (22-24), even as the bulk PIP2 mass is reduced. The equally attractive possibility that PIP2 binding is regulated by signals generated during agonist stimulation has not been considered.

Agonist-stimulated cells exhibit complex Ca2+ oscillations and pH transients. These signals alter the binding of gelsolin and CapG to actin, by inducing a conformational change (6, 25-27). In this study, we tested the effect of Ca2+ and pH on the binding of the gelsolin family proteins to PIP2 and found that they affect PIP2 binding in an interdependent manner. We identified the domains in gelsolin that impart such regulation and elucidated the relation between the NH2-terminal and COOH-terminal halves of the protein. Since gelsolin modulates the activity of many PIP2-regulated proteins with important signaling functions in vivo (28) and in vitro (29-31), our results have important implications for how the gelsolin family proteins are regulated during agonist signaling and how the activity of other PIP2-dependent cytoskeletal and noncytoskeletal proteins can be coordinated.


EXPERIMENTAL PROCEDURES

Expression and Purification of Recombinant CapG, Gelsolin, and Gelsolin Domains

Gelsolin has six semihomologous domains (S1-6), which can be further divided into two functional halves (32). The expression vectors for the gelsolin NH2-terminal half (S1-3), gelsolin S1, gelsolin S2-3, and CapG have been described previously (33-35). The full-length gelsolin expression vector (encompassing the entire human plasma gelsolin coding sequence) was constructed by ligating gelsolin cDNA to pet3a via the BamHI site. Recombinant proteins were expressed in bacteria and purified using sequential anion and cation exchange chromatography (34). Protein concentration was determined by the method of Bradford (36), and protein purity was assessed by SDS-polyacrylamide gel electrophoresis.

The COOH-terminal half expression vector was constructed by using polymerase chain reaction to generate a fragment encompassing human plasma gelsolin nucleotides 1298-1753. The forward primer contains a XhoI site (ACC TCC ACT CTC GAG GCC GCC), and the reverse primer has a SmaI site (CAA CAG CCC GGG TGG CT). The polymerase chain reaction product was cloned into Bluescript KS+ via the XhoI/SmaI sites. This construct was digested with SmaI and blunt end-ligated with a downstream gelsolin fragment. The fragment was excised with BamHI from full-length gelsolin cDNA in Bluescript KS+ (gelsolin SmaI site at nucleotide 1750 and vector multiple cloning SmaI site downstream of the termination codon). The resultant cDNA was digested with SpeI (in the 3' multiple cloning region, downstream of SmaI) and filled in with CT nucleotides to create a site with a two-base overhang compatible with that of HindIII. The other end was released by digestion with XhoI and ligated to PGEX K6 vector that was linearized with HindIII (site partially filled in with nucleotides AG to generate a two-base overhang compatible with the partially filled in SpeI) and XhoI. The fusion protein contained a 30-kDa GST followed by a 40-kDa gelsolin COOH-terminal half. The COOH-terminal gelsolin was cleaved from GST bound to a column with thrombin.

Phospholipid

PIP2 was purchased from Calbiochem. Micelles were prepared by dissolving the dried lipid in water to a final concentration of 2 mg/ml and sonicating for 5 min. at maximum power (model W185; Heat Systems Ultrasonics, Inc., Farmingdale, NY). Large unilamellar vesicles at a 5:1 phosphatidylcholine:PIP2 ratio were made with an extruder (Lipex Biomembranes, Vancouver, Canada) as described by Machesky et al. (37).

Small Zone Gel Filtration

The assay was similar to that described previously for studying lipid binding to most actin regulatory proteins (33, 35, 38). This is because small proteins bound to PIP2 micelles or mixed vesicles migrate faster than the unbound proteins. Proteins were incubated with lipid for 30 min at room temperature, and 100 µl of the mixture was chromatographed at 4 °C through a Superdex 75 HR 10/30 column (Pharmacia Biotech Inc.), equilibrated with pH 7.0 or 7.5 buffers containing 25 mM Hepes, 100 mM KCl, 0.5 mM beta -mercaptoethanol, 0.4 mM EGTA with or without CaCl2. Lipid was not included in the elution buffer. Fractions were eluted at 0.5 ml/min, and 0.5-ml fractions were collected. The elution profile was monitored by absorbance at 280 nm. The amount of unbound protein was determined from the protein absorbance peak. The lipid-bound protein was calculated as the difference between the total protein applied minus the unbound protein. The apparent dissociation constant (Kd) was calculated as follows.
K<SUB>d</SUB>=[<UP>protein</UP>]<SUB><UP>free</UP></SUB>×[<UP>lipid</UP>]<SUB><UP>free</UP></SUB>/[<UP>protein-lipid</UP>] (Eq. 1)

Equilibrium Gel Filtration

The method of Hummel and Dreyer (39), as modified by Machesky et al. (37) was used. A Superose 12 HR 10/30 column (Pharmacia) was equilibrated with CapG (ligand) in a buffer containing 25 mM Hepes, 75 mM KCl, 0.5 mM dithiothreitol, 1.8 mM NaN3, 0.05 µM CaCl2, pH 7.5, at room temperature. 100 µl of the equilibration buffer containing CapG was incubated with PIP2 micelles for 30 min and loaded onto the column. The column was developed with the equilibration buffer containing CapG at 0.25 ml/min, and 0.3-ml fractions were collected. CapG concentration in the column fractions was monitored by UV absorption. The amount of CapG bound to PIP2 was determined from the trough in the absorbance peak. Multiple runs using equilibration CapG concentrations of 0.96, 1.3, 2.6, and 3.9 µM and PIP2 concentrations of 34, 46, 68, and 91 µM were done. Kd was determined by the equation,
r=<FR><NU>B<SUB><UP>max</UP></SUB>[<UP>protein</UP>]<SUB><UP>free</UP></SUB></NU><DE>K<SUB>d</SUB>+[<UP>protein</UP>]<SUB><UP>free</UP></SUB></DE></FR> (Eq. 2)
where r is the ratio of protein bound to each PIP2 molecule at a given PIP2 concentration and Bmax is the maximum number of protein bound per PIP2 at saturation.

Quenching of Intrinsic Tryptophan Fluorescence

Fluorescence spectra were recorded at 30 °C with a QM-1 fluorometer (Photon Technology International, Canada). 2 ml of a protein solution (0.3 µM, 30 °C) in 25 mM Hepes, 100 mM KCl, 0.4 mM EGTA, 0.5 mM beta -mercaptoethanol, pH 7.5, with or without 36 µM free Ca2+ were placed in a 1-cm square quartz cuvette and stirred with a minimagnetic stirrer. After allowing 5 min for equilibration, the tryptophan fluorescence spectrum was recorded by excitation at 292 nm. The excitation and emission beam slits were set at 3 and 2 nm bandwidth, respectively. PIP2 micelles (at final PIP2 concentrations ranging from 0.042 to 32.3 µM, depending on the protein studied) were added at 2-µl increments, and the fluorescence spectra were recorded 5 min after each addition. The total volume of micelles added did not exceed 2% of the initial protein solution volume. The decrease in fluorescence emission at 320 nm was plotted as a function of PIP2 concentration, and the fluorescence change was assumed to be proportional to the concentration of the protein-phosphoinositide complex. Data were analyzed as described by Ward (40). The apparent dissociation constant, Kd, was calculated using the equation,
&Dgr;F=(&Dgr;F<SUB><UP>max</UP></SUB>×[<UP>lipid</UP><SUB>T</SUB>])/(K<SUB>d</SUB>+[<UP>lipid</UP><SUB>T</SUB>]) (Eq. 3)
where Delta F is the fluorescence quenching at a given PIP2 concentration, Delta Fmax is the total fluorescence quenching of the protein saturated with ligand, and [lipidT] is the concentration of PIP2. Delta Fmax is estimated by curve fitting of the binding data using the Hyperbol.fit program in SigmaPlot. Alternatively, the intrinsic association constant (Ka) as well as the stoichiometry of binding (p) can be derived using the graphical method of Stinson and Holbrook (41),
<FR><NU>1</NU><DE>(1−&thgr;)K<SUB>a</SUB></DE></FR>=<FR><NU>[<UP>lipid</UP><SUB>T</SUB>]</NU><DE>&thgr;</DE></FR>−p[<UP>protein</UP><SUB>T</SUB>] (Eq. 4)
where theta  is the fractional binding (Delta F/Delta Fmax), p is the stoichiometry of binding, [lipidT] is the total concentration of PIP2, and [proteinT] is the total acceptor concentration. When 1/(1 - theta ) is plotted against lipidT/theta , a straight line with a slope of Ka and an intercept of proteinT/theta is obtained. The stoichiometry of interaction (p) can be calculated by dividing the intercept with the protein concentration.

Measurement of Free Ca2+ Concentration and pH

The concentrations of free Ca2+ in EGTA containing solutions with varying amounts of Ca2+ were measured with Ca2+-sensitive dyes. 5 µM Fura-2 was used to determine Ca2+ concentrations below 1 µM. Free Ca2+ concentration was calculated (26) assuming the Kd of the Fura-2-Ca2+ complex is 229 nM at pH 7.0 and 144 nM at pH 7.5. Calcium green 5N (Molecular Probes, Eugene, OR) was used to measure Ca2+ concentrations higher than 1 µM, and free Ca2+ concentration was calculated assuming a Kd of 14 µM.


RESULTS

CapG Binding to PIP2

Small zone gel filtration analyses showed that CapG bound to PIP2 micelles in a dose-dependent manner. Micelle-bound CapG eluted in the void volume that was well separated from the free protein peak (Fig. 1). Binding to phosphatidylcholine-PIP2 vesicles gave similar results (data not shown), suggesting that micelles could be used to assess binding, although it is not a physiological substrate. To facilitate comparison under different binding conditions and between different proteins, we attempted to calculate a Kd. Equilibrium binding studies suggest that each CapG binds two PIP2 molecules (see below). Assuming this stoichiometry, the apparent Kd for binding to PIP2 micelles (calculated using Equation 1) was 69.0 µM in 1 mM EGTA, and 29.4 µM in the presence of 36 µM Ca2+ at pH 7.5 (Table I). These values represent the upper limit, since measurements were not made under equilibrium conditions.


Fig. 1. Small zone gel filtration of CapG. A-F, elution profiles of CapG with increasing PIP2 concentration. CapG (5.5 µM) was incubated with PIP2 micelles in a pH 7.5 buffer containing 0.4 mM EGTA. PIP2 concentrations were 0, 14.2, 22.7, 28.3, 56.7, and 113.3 µM for A-F, respectively.
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Table I. Binding of CapG to PIP2

Kd for fluorescence titration was calculated using Equation 4. Kd values for gel filtration data were calculated with Equation 1, assuming a stoichiometry of 2. Values shown are mean ± S.E., determined at pH 7.5. 

KdM)
Fluorescence
Gel filtration
EGTA Ca2+ pa hb Small zone
Equilibrium (Ca2+)
EGTA Ca2+

24.4 ± 5.9 (n = 3)c 6.0 ± 0.8 (n = 3) 1.7 1.1 ± 0.03 (n = 3) 69.0 ± 5.3 (n = 5) 29.4 ± 2.5 (n = 7) 7.0 ± 0.5 (n = 5)

a p, mol of PIP2/mol of CapG, average of two determinations.
b h, Hill coefficient.
c n, number of independent experiments.

To determine if there is indeed a Ca2+-induced change, equilibrium binding studies based on the quenching of CapG intrinsic tryptophan fluorescence by PIP2 were performed. This method has been used to study the binding of profilin (43), phospholipase Cdelta (44), and dynamin pleckstrin homology domain (45) to PIP2. CapG had an emission maximum of 327 nm, and 36 µM Ca2+ produced a small reduction in fluorescence intensity (the ratio of peak fluorescence in EGTA/Ca2+ is 0.92 ± 0.05 (mean ± S.E., n = 5) (Fig. 2, A and B). PIP2 induced a dose-dependent and saturable decrease in intrinsic fluorescence, without shifting the emission maximum. Micelles alone without CapG did not have significant emission (data not shown). A plot of CapG fluorescence quenching versus PIP2 concentration showed that saturation was reached at a lower PIP2 concentration in the presence of Ca2+ than in EGTA (Fig. 3A). The Kd values for binding at pH 7.5, calculated according to Equation 3, were 31.9 and 8.4 µM in EGTA and Ca2+, respectively, for the experiment shown. Similar values (24.4 and 6.0 µM) were obtained when the data were analyzed using Equation 4 (Table I). These Kd values were 3-4 times lower than the small zone gel filtration values, suggesting that CapG-PIP2 complexes dissociate during nonequilibrium gel filtration. Using a similar protocol, human platelet profilin binds PIP2 with a Kd of 35 µM (43), and binding is not affected by Ca2+.


Fig. 2. Tryptophan fluorescence emission spectra of CapG. 2 µl of 0.85 mM PIP2 micelles were added sequentially to CapG in the absence or presence of 36 µM free Ca2+. A, emission curves of 0.3 µM CapG in a pH 7.5 buffer in the presence of 0.4 mM EGTA. The top curve was without PIP2, and the remaining curves were with PIP2 ranging from 0.85 to 32.3 µM. B, emission curves in the presence of 36 µM free Ca2+ and 0-32 µM PIP2, as described in A.
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Fig. 3. Analysis of CapG fluorescence titration data. A, binding curves plotting (Fmax - F) versus PIP2 concentration. Closed and open circles represent experimental points in 0.4 mM EGTA or 36 µM Ca2+. Dotted lines are fitted to the experimental points, and Fmax values were obtained. Kd values with and without Ca2+ were 8.4 and 31.9 µM, respectively, for this experiment. B, Hill plot of the titration data. The Hill equation is rearranged and plotted to show the relation between the log(Y/(1 - Y)) and log[lipidT], where Y is the fractional saturation. h, the Hill coefficient, is derived from the slope (1.1 and 0.97 in the presence of Ca2+ and EGTA for this experiment).
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The stoichiometry of CapG binding was 1.7 in either Ca2+ or EGTA (Table I). Since CapG has one known PIP2 binding site (6, 33), this site appears to bind two PIP2 molecules. The two PIP2 bound independently and noncooperatively, as indicated by the Hill coefficients of close to 1 (1.02 ± 0.05 and 1.09 ± 0.02 in EGTA and Ca2+, respectively) (Fig. 3B, Table I). The exact meaning of this stoichiometry is not clear, because each micelle contains multiple PIP2 and CapG can potentially bind more than one micelle. Nevertheless, the calculated stoichiometry is useful for comparison among different proteins.

Equilibrium gel filtration validated the Kd derived by fluorescence titration. The column was preequilibrated with CapG, and PIP2 incubated with CapG in the equilibrating buffer was added. The column was then developed with CapG containing equilibration buffer. CapG bound to PIP2 migrated faster, increasing the CapG content above the equilibration level (peak) and depleting the amount in the trailing fractions (trough) (Fig. 4, A and B). Assuming that each CapG bound two PIP2 molecules (see Table I), the Kd obtained from five experiments performed with a range of CapG and/or PIP2 concentration was 8.1 ± 0.9 µM (mean ± S.E.). This is comparable with the spectroscopic titration result, affirming the validity of the two independent methods.


Fig. 4. Hummel-Dyer equilibrium gel filtration to characterize CapG binding to PIP2 micelles. A, elution profile. The column was equilibrated with 1.4 µM CapG, and 68 µM CapG was added. B, Coomassie Blue staining of CapG in column fractions. 50-µl aliquots were lyophilized and analyzed by SDS-polyacrylamide gel electrophoresis.
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Gelsolin Binding to PIP2

Tryptophan titration could not be used to study gelsolin binding to PIP2 because the full-length gelsolin signal (without phosphoinositide) fluctuated and did not reach a steady level even after 20 min. The reason for this instability was not investigated further. Gel filtration experiments showed that gelsolin binding to PIP2 was enhanced by Ca2+ (Fig. 5, A-F). At pH 7.5, the apparent Kd values were 305.4 and 40.2 µM with and without Ca2+ (Table II). The latter value is similar to that of CapG, indicating that gelsolin and CapG have comparable PIP2 binding affinity in the presence of Ca2+. However, in EGTA, gelsolin has a much higher Kd than CapG, suggesting that Ca2+ induces a larger change in binding affinity. This could be due to a disproportionate increase in koff relative to kon. 10 µM Mg2+ did not substitute for Ca2+ (data not shown), consistent with previous results (46).


Fig. 5. Effects of Ca2+ and pH on the binding of gelsolin to PIP2, as determined by small zone gel filtration. 2.3 µM gelsolin was incubated with 56.7 µM PIP2 in a pH 7.5 or 7.0 buffer containing 0.4 µM EGTA and increasing amounts of CaCl2. The free Ca2+ concentration was determined as described under "Experimental Procedures." A-F, gelsolin elution profiles. A-C, pH 7.5; D-F, pH 7.0. The Kd values at pH 7.5, from left to right, are 323.2, 298.6, and 118.6 µM, respectively. Kd values at pH 7.0, from left to right, are 345.2, 119.7, and 16.2 µM, respectively. G, plot of Kd versus Ca2+ concentration, at two different pH values.
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Table II. Binding of gelsolin to PIP2


KdM)
Fluorescencea
Gel filtration
EGTA Ca2+ pb hc EGTA Ca2+

Gelsolin (S1-6) (3.0) 305.4  ± 20.2 (n = 7)d 40.2  ± 10.7 (n = 3)
NH2-half (S1-3) 1.3  ± 0.3 2.9  ± 0.7 3.4 1.0  ± 0.03 4.3  ± 1.1 (n = 7) 7.4  ± 0.7 (n = 4)
  S1 4.2  ± 2.3 1.6 0.9  ± 0.2
  S2-3 1.0  ± 0.2 2.9  ± 0.9 2.1 1.0  ± 0.1
COOH-half (S4-6) 9.7  ± 0.4 20.0  ± 1.2 0.9  ± 0.2 52.0  ± 3.0 (n = 3) 137.7  ± 4.2 (n = 3)

a Fluorescence titration results were mean ± S.E. for three independent experiments.
b p, stoichiometry of binding (mol of PIP2/mol of protein), average of two determinations. Stoichiometry for gelsolin is assumed to be 3.
c h, Hill coefficient.
d n, number of gel filtration experiments.

The effect of Ca2+ was amplified when the pH was shifted from 7.5 to 7.0 (Fig. 5, compare A-C with D-F). The relations among Kd, Ca2+, and pH are shown in Fig. 5G. In the absence of Ca2+, decreasing pH from 7.5 to 7.0 had minimal effect (Kd of 300 and 350 µM, respectively). This is not surprising, since PIP2 protonation is not expected to change substantially within this narrow pH range (47) and a broader pH range does not affect binding of profilin to PIP2 either (37). However, at pH 7.0, less Ca2+ was required to increase binding. 0.2 µM Ca2+ decreased the Kd by half at pH 7.0, while 4.5 µM Ca2+ was required to produce the same effect at pH 7.5. Both Ca2+ concentrations are well within the range achieved following agonist stimulation, particularly at the cytoplasm immediately subjacent to the plasma membrane.

Ca2+ and pH Regulation of Gelsolin Domains

To determine which part of gelsolin contributes to the Ca2+ and/or pH dependence of PIP2 binding, we examined the PIP2-binding characteristics of several gelsolin domains. Gelsolin contains six segmental repeats, S1-6 (32). The NH2-terminal half encompassing S1-3 binds actin independently of Ca2+ (48) and has two known PIP2 binding sites and potentially a third unmapped site (33, 49, 50). The COOH-terminal half (S4-6), which requires Ca2+ to bind actin (51), has not been examined previously for PIP2 binding.

Unlike full-length gelsolin, the gelsolin NH2-terminal half behaved well during fluorescence titration (Fig. 6A). It bound PIP2 with high affinity, and saturation was reached at a slightly lower PIP2 concentration in EGTA than in Ca2+ (the opposite of full-length gelsolin and CapG). The Kd values for the experiment shown in Fig. 6A were 1.2 and 2.9 µM, respectively. The stoichiometry of binding, derived from Fig. 6B, was 3.4. This value is twice that of CapG, confirming that gelsolin NH2-terminal half has more PIP2 binding sites (33). Gel filtration studies confirmed that Ca2+ increased the Kd. The Hill coefficient of 1.1 ± 0.03 (Fig. 6C, Table II) suggested that binding was noncooperative and that the sites bound PIP2-independently. S1, which has one PIP2 site, bound 1.6 mol of PIP2 with a Kd of 4.2 µM in EGTA, while S2-3 bound 2.1 µmol of PIP2 with a Kd of 1.0 and 2.9 µM in EGTA and Ca2+, respectively (Table II).


Fig. 6. Effects of Ca2+ on the binding of the gelsolin NH2-terminal half to PIP2, as determined by fluorescence titration. A, NH2-terminal half (0.2 µM), with and without 36 µM Ca2+ in buffer containing 0.4 mM EGTA. PIP2 concentration ranged from 0.04 to 10.2 µM. B, binding curves as described by Stinson and Holbrook (41). Ka is derived from the slope, and p, the stoichiometry of binding, is derived from the intercept divided by the protein concentration. C, Hill plot of binding in the presence of EGTA.
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The gelsolin COOH-terminal half bound PIP2 with much lower affinity (approximately 7-fold higher Kd by fluorescence measurements) than the NH2-terminal half (Table II). It is therefore probably not involved in PIP2 binding per se. As with the NH2-terminal half, binding to the COOH-terminal half was reduced in Ca2+ (Fig. 7C). This is in sharp contrast to the large Ca2+-enhancement of PIP2 binding to full-length gelsolin. The opposite effects of Ca2+ on full-length and half-length gelsolins therefore cannot simply be due to nonspecific lipid aggregation. The pronounced enhancement of PIP2 binding to full-length gelsolin most likely reflects a Ca2+-dependent exposure of the NH2-terminal half PIP2 binding sites through a change in the COOH-terminal half. This conclusion is based on the observation that neither the NH2- nor COOH-terminal halves are activated by Ca2+ to bind PIP2, and only the COOH-terminal half is known to undergo Ca2+-induced conformational change (51).


Fig. 7. Effects of pH on the binding of gelsolin NH2- and COOH-terminal halves to PIP2, as determined by gel filtration. A, gelsolin NH2-terminal half (4 µM) binding to 11.3 and 22.7 µM PIP2 as a function of pH. Values shown are the mean of two determinations, and the range is indicated. B, COOH-terminal half (5.8 µM) binding to 22.7 µM PIP2 as a function of pH and Ca2+.
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Gelsolin NH2-terminal half binding to PIP2 was enhanced by lowering pH. The Kd dropped from 8.2 to 3.4 µM between pH 7.5 and 7.0 in the presence of EGTA (Fig. 7A). In contrast, the gelsolin COOH-terminal half was not affected by pH (Fig. 7B).


DISCUSSION

Actin polymerization in response to agonist activation is frequently associated with a rise in cytosolic Ca2+, changes in PIP2 content, and intracellular pH. There is also compelling evidence that gelsolin, which severs and caps actin filaments in response to changes in Ca2+ and PIP2 concentration and pH, is involved in actin remodeling (17, 52-54). In this paper, we show that gelsolin and CapG binding to PIP2 is affected by physiologically relevant changes in Ca2+ and pH. The effects are not due to alterations in PIP2 structure per se but reflect changes in the proteins. This is the first report that PIP2 binding to any protein is directly modulated by signals generated during agonist stimulation and has implications for divergent PIP2-dependent processes beyond a direct effect on the cytoskeleton.

The finding that gelsolin binding to PIP2 is promoted by Ca2+ is consistent with the current model for how gelsolin is activated by Ca2+ to bind actin (48, 51). Our deletion studies suggest that the extreme COOH terminus of gelsolin is critical to the inhibition of the NH2-terminal actin binding sites, because gelsolin lacking the COOH-terminal 23 residues no longer requires Ca2+ to bind actin (56). We do not know at present whether actin binding and PIP2 binding are regulated identically. This question can now be addressed, because the actin and PIP2-binding sites of gelsolin have been mapped (33, 50, 56-58) and the crystal structures of gelsolin S1 complexed with actin (57) and full-length gelsolin in EGTA2 have been solved recently.

Less is known about how pH affects gelsolin conformation. Selve and Wegner (59) first reported that pH 6 increases the rate of gelsolin binding to actin in the presence of Ca2+. Lamb et al. (26) subsequently showed that the Ca2+ requirement for gelsolin severing is reduced at pH 6.5 and abolished at pH below 6.0. pH 5 induces gelsolin unfolding, as determined by dynamic light scattering (26). We find that a less extreme pH drop potentiates Ca2+ activation of PIP2 binding to full-length gelsolin. Acidic pH increases the NH2-terminal half binding to PIP2 even without Ca2+ but has no effect on COOH-terminal half binding. Therefore, mild acidification probably promotes PIP2 binding by directly altering the NH2-terminal PIP2 binding sites.

The significance of an increase in PIP2 affinity described here depends on the PIP2 concentration in the plasma membrane. This is difficult to estimate precisely because PIP2 may be partitioned and sequestered. One estimate, based on PIP2 accounting for 1% of plasma membrane lipid, suggests that the PIP2 concentration in the plasma membrane of a spherical cell with a radius of 10 µm is 10 µM (44). In platelets, the PIP2 concentration is estimated to be about 300 µM when averaged over the entire cell volume (internal and plasma membranes) (60), and PIP2 concentration decreases by 30% following stimulation (16). Cytosolic [Ca2+] rises during agonist stimulation, and the 4-8-fold increase in CapG and gelsolin binding affinity described here is sufficiently large to promote their increased association with the plasma membrane despite a modest decrease in membrane PIP2. The magnitude of the increase depends on the PIP2 concentration before and after stimulation. Immunogold labeling studies show that 4 and 6.5% of gelsolin is associated with the plasma membrane in resting and activated platelets, respectively (42). This represents a 63% increase in membrane association after stimulation. Our finding that Ca2+ increases PIP2 binding affinity can explain how PIP2 uncaps gelsolin and CapG even as the plasma membrane PIP2 content decreases following agonist stimulation.

Since only a handful of the currently identified PIP2-binding proteins are Ca2+- and pH-sensitive, our finding is consistent with a selective regulation of the gelsolin family. Nevertheless, increased gelsolin and CapG binding will impact multiple PIP2-dependent processes by altering PIP2 availability to other binding proteins, especially when PIP2 concentration is decreased during agonist stimulation. Some actin-binding proteins are inhibited by PIP2 (profilin, cofilin, capping protein), while others are activated (alpha -actinin and vinculin). Gelsolin and CapG can therefore exert positive as well as negative effects indirectly by controlling PIP2. We postulate that as the cytosolic [Ca2+] rises during stimulation, gelsolin severs filaments and PIP2 dissociates it from the filament end. Increased gelsolin binding to PIP2 displaces capping protein and profilin, neither of which are Ca2+-sensitive, from the plasma membrane. Profilin catalyzes polymerization (16), and the reaction is terminated by capping protein-mediated filament capping (55). Multiple rounds of severing, uncapping, and facilitated actin addition at the barbed ends fuel explosive amplification of filament growth observed during lamellipodial extension and membrane ruffling.

Our findings also have implications beyond a direct effect on the cytoskeleton. Many important signaling proteins are regulated by PIP2 as well. It is significant that several pleckstrin homology proteins (reviewed in Ref. 2) bind PIP2 with similar affinity as the gelsolin family. For example, the Kd values of beta -adrenergic receptor kinase type 1, pleckstrin, dynamin, and phospholipase Cdelta are 50, 50, 4, and 1 µM, respectively. Therefore, gelsolin and CapG can potentially compete with them for PIP2, particularly when the [Ca2+] rises and PIP2 level drops during agonist stimulation. This possibility is supported by in vitro and in vivo experiments. In vitro, gelsolin stimulates and inhibits inositol-specific phospholipase C isozymes in a biphasic manner (29).3 Gelsolin stimulates phosphoinositide 3-OH-kinase (31), although we find that gelsolin and CapG also inhibit it.4 Gelsolin activates phospholipase D (30) in a PIP2-dependent manner. Modest overexpression of CapG (28) or gelsolin has profound effects on phospholipase Cbeta and phospholipase Cgamma activated through two distinct receptor-mediated pathways.3

In conclusion, these observations show that gelsolin and CapG binding to PIP2 is selectively regulated by second messengers. This regulation provides an additional level of control above that of a bulk change in PIP2 content. Differential modulation and cross-talk between the PIP2-binding proteins allow control to be exerted at multiple points in the signaling cascade.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants RO1 GM5112 (to H. L. Y.) and GM53448 (to C. S. C.) and individual National Research Service Award GM17997 (to E. 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.
   To whom correspondence should be addressed: Dept. of Physiology, U.T. Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214-648-7967; Fax: 214-648-8685; E-mail: yin01{at}utsw.swmed.edu.
1   The abbreviation used is: PIP2, phosphatidylinositol 4,5-bisphosphate.
2   Burtnick, L. D., Koepf, E. K., Grimes, J., Jones, E. Y., Stuart, D. I., McLaughlin, P. J., and Robinson, R. C. (1997) Cell, in press.
3   Sun, H.-Q., Lin, K.-M., and Yin, H. L. (1997) J. Cell Biol., in press.
4   P.-J. Lu, A.-L. Hsu, D.-S. Wang, H. Yan, H. L. Yin, and C.-S., Chen, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Drs. J. Albanesi, D. Hilgemann, and P. Thomas for helpful discussions and L. Segura for excellent technical assistance.


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