Membrane Targeting of C2 Domains of Phospholipase C-δ Isoforms

The C2 domain is a Ca2+-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-δ isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-δ1, -δ3, and -δ4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-δ1-C2 is driven by nonspecific electrostatic interactions between the Ca2+-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca2+, Asn647, and phosphatidylserine (PS). The PS selectivity of PLC-δ1-C2 governs its specific Ca2+-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-δ3-C2 also involves Ca2+-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-δ1-C2 and PLC-δ3-C2, PLC-δ4-C2 has significant Ca2+-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca2+-binding loops and the substitution of Ser for the Ca2+-coordinating Asp in position 717. Consequently, PLC-δ4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca2+ import and non-selective Ca2+-mediated targeting to various cellular membranes, suggesting that PLC-δ4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-δ isoforms as Ca2+-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors. The C2 domain is a Ca2+-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-δ isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-δ1, -δ3, and -δ4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-δ1-C2 is driven by nonspecific electrostatic interactions between the Ca2+-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca2+, Asn647, and phosphatidylserine (PS). The PS selectivity of PLC-δ1-C2 governs its specific Ca2+-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-δ3-C2 also involves Ca2+-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-δ1-C2 and PLC-δ3-C2, PLC-δ4-C2 has significant Ca2+-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca2+-binding loops and the substitution of Ser for the Ca2+-coordinating Asp in position 717. Consequently, PLC-δ4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca2+ import and non-selective Ca2+-mediated targeting to various cellular membranes, suggesting that PLC-δ4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-δ isoforms as Ca2+-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors. phospholipases C bovine serum albumin cytosolic phospholipase A2 Dulbecco's modified Eagle's medium enhanced green fluorescence protein fetal bovine serum glutathione S-transferase inositol 1,4,5-trisphosphate phosphatidylcholine phosphatidylglycerol protein kinase C 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine phosphatidylserine 5)P2, phosphatidylinositol 4,5-bisphosphate surface plasmon resonance 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid nickel-nitrilotriacetic acid Mammalian phosphatidylinositol-specific phospholipases C (PLC)1 are responsible for converting phosphatidylinositol 4,5-bisphosphate (Ins(4,5)P2) into diacylglycerol and inositol 1,4,5-trisphosphate (IP3), which promote the activation of protein kinases C (PKC) and the release of Ca2+ from intracellular stores, respectively (1Williams R.L. Biochim. Biophys. Acta. 1999; 1441: 255-267Crossref PubMed Scopus (93) Google Scholar, 2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1212) Google Scholar). The PLC family comprises eleven isoforms that can be subdivided into four types (β, γ, δ, and ε) based on their structural differences. All PLC isoforms except newly discovered PLC-ε possess three regulatory domains: PH, EF-hand, and C2 (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1212) Google Scholar). Among PLC isoforms, Ca2+-sensitive PLC-δ1 has been the subject of extensive structure-function studies due to the availability of tertiary structural information. Crystallographic structures of PLC-δ1 lacking the amino-terminal PH domain (3Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar) and of its isolated PH domain (4Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (528) Google Scholar) revealed a four-module organization of the enzyme comprising the amino-terminal PH domain, the EF-hand domain, catalytic domain, and the carboxyl-terminal C2 domain. Based on these structures, it was proposed that PH and C2 domains, both of which are well-characterized membrane-targeting domains, are involved in the membrane targeting of PLC-δ1 (3Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar). The PH domain is a β-barrel-like structure that is present in many membrane-binding proteins (5Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 6Blomberg N. Baraldi E. Nilges M. Saraste M. Trends Biochem. Sci. 1999; 24: 441-445Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (249) Google Scholar). The essential role of the PH domain in the membrane targeting of PLC-δ1 has been experimentally demonstrated both in vitro and in vivo (8Yagisawa H. Sakuma K. Paterson H.F. Cheung R. Allen V. Hirata H. Watanabe Y. Hirata M. Williams R.L. Katan M. J. Biol. Chem. 1998; 273: 417-424Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The PH domain of PLC-δ1 is capable of anchoring the protein to the membrane by specifically binding to Ins(4,5)P2 in the membrane, and the competitive binding of the PH domain to soluble IP3 can induce the membrane dissociation of PLC-δ1. However, the role of the C2 domain in PLC-δ1 catalysis remains unclear. The C2 domain has been identified in many cellular proteins involved in signal transduction or membrane trafficking (9Nalefski E.A. Slazas M.M. Falke J.J. Biochemistry. 1997; 36: 12011-12018Crossref PubMed Scopus (113) Google Scholar, 10Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 11Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Many C2 domains bind Ca2+ and mediate Ca2+-dependent membrane targeting of proteins. Structural analyses of multiple Ca2+-dependent membrane-binding C2 domains have shown that they share a common fold consisting of an eight-stranded antiparallel β-sandwich connected by variable loops, which form the binding sites for multiple Ca2+ ions at one end of the domain (3Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar, 12Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar, 13Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 14Shao X. Fernandez I. Sudhof T.C. Rizo J. Biochemistry. 1998; 37: 16106-16115Crossref PubMed Scopus (203) Google Scholar, 15Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (283) Google Scholar, 16Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 17Xu G.Y. McDonagh T. Yu H.A. Nalefski E.A. Clark J.D. Cumming D.A. J. Mol. Biol. 1998; 280: 485-500Crossref PubMed Scopus (100) Google Scholar). The crystal structure of the PLC-δ1 C2 domain reveals three metal binding sites in the loop region (18Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar, 19Grobler J.A. Essen L.O. Williams R.L. Hurley J.H. Nat. Struct. Biol. 1996; 3: 788-795Crossref PubMed Scopus (102) Google Scholar), which led to the proposal that the C2 domain is involved in the Ca2+-dependent membrane targeting of the protein. However, a mutant of PLC-δ1 lacking the C2 domain Ca2+ binding sites showed the same activity toward Ins(4,5)P2 in phosphatidylcholine (PC)-containing vesicles and micelles as the wild type, suggesting the Ca2+ requirement of PLC-δ1 largely reflects the binding of Ca2+ to the active site (20Grobler J.A. Hurley J.H. Biochemistry. 1998; 37: 5020-5028Crossref PubMed Scopus (46) Google Scholar). More recently, it was shown that, in the presence of phosphatidylserine (PS) in the assay mixture, the C2 domain plays a key role in the activation of PLC-δ1 through the formation of a C2·Ca2+·PS ternary complex (21Lomasney J.W. Cheng H.F. Roffler S.R. King K. J. Biol. Chem. 1999; 274: 21995-22001Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To better understand the role of the C2 domain in the membrane targeting and activation of PLC-δ1 and other isoforms, we measured the binding of the C2 domains of PLC-δ1, -δ3, -δ4, and their mutants to model membranes, and analyzed the binding in terms of the electrostatic properties of the domains. We also measured the spatiotemporal dynamics of enhanced green fluorescence protein (EGFP)-tagged C2 domains and mutants in living cells. Results described herein establish the C2 domains of PLC-δ isoforms as Ca2+-dependent membrane targeting domains that have distinct membrane binding properties, which in turn control their subcellular localization behaviors. Together, these studies shed new light on the roles of the C2 domains in the membrane targeting and activation of PLC-δ isoforms. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Ins(4,5)P2 was from Calbiochem (San Diego, CA). Tritiated Ins(4,5)P2([3H]Ins(4,5)P2) was purchased from PerkinElmer Life Sciences (Boston, MA). Phospholipid concentrations were determined by phosphate analysis (22Kates M. Techniques of Lipidology.2nd Ed. Elsevier, Amsterdam1986: 114-115Google Scholar). The Liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario, Canada). Fatty acid-free bovine serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). Triton X-100 was obtained from Pierce Chemical Co (Rockford, IL). Restriction endonucleases and enzymes for molecular biology were obtained from either Roche Molecular Biochemicals or New England BioLabs (Beverly, MA). CHAPS and octyl glucoside were from Sigma Chemical Co. and Fisher Scientific, respectively. Pioneer L1 sensor chip was from BIAcore AB (Piscataway, NJ). Ionomycin was from Calbiochem (San Diego, CA). Zeocin and ponasterone A were from Invitrogen (Carlsbad, CA). To subclone the cDNA of PLC-δ1 into the pGEX-4T-1 vector (Amersham Biosciences, Inc., Piscataway, NJ) that contains the amino-terminal glutathione S-transferase (GST) sequence, the start codon was removed and new restriction sites (SmaI andXhoI) were constructed in the gene by overlap extension PCR mutagenesis using Pfu polymerase (Stratagene, La Jolla, CA). Using NdeI and HindIII sites, the cDNAs of PLC-δ3 and PLC-δ4 were subcloned into the pET28a vector (Novagen, Madison, WI) that encodes the amino-terminal His6 tag and thrombin cleavage site (MGSSHHHHHHSSGLVPRGSH). The isolated C2 domains of all three isoforms were subcloned similarly into the pET28a vector. Mutants were generated by overlap extension PCR mutagenesis. Escherichia coli strain BL21 (for pGEX-4T-1 vector) and BL21(DE3) (for pET28a vector) were used as hosts for protein expression. One liter of Luria broth supplemented with 100 μg/ml ampicillin for PLC-δ1 and 50 μg/ml kanamycin for PLC-δ3 and PLC-δ4 was inoculated with 1 ml of overnight culture grown at 37 °C. Cells were grown at 37 °C until the absorbance at 600 nm reached ∼0.6, then the protein expression was induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside (Research Products, Mount Prospect, IL). After overnight incubation at room temperature, cells were harvested by centrifugation at 5000 ×g for 10 min at 4 °C. Cells were resuspended in 50 ml of 50 mm Tris-HCl buffer, pH 8.0, containing 50 mmNaCl, 2 mm EDTA, 0.4% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, and 1 mm phenylmethylsulfonyl fluoride, and sonicated. The supernatant was collected by centrifugation at 50,000 × g for 20 min at 4 °C. His-tagged proteins (PLC-δ3 and PLC-δ4) were purified using a Ni-NTA-agarose column according to the manufacturer's instructions. The GST fusion protein (PLC-δ1) was purified using a glutathione-Sepharose affinity system as follows. The cell lysate was incubated with GST beads for 1 h at 4 °C. The unbound protein impurities were washed with 50 mm Tris-HCl buffer, pH 8.0, containing 150 mm NaCl. The GST-fused protein still bound to the beads was incubated with thrombin for 6 h at 25 °C. Then, the cleaved protein was eluted with the same buffer. Isolated C2 domains were prepared as follows. One liter of Luria broth supplemented with 50 μg/ml kanamycin was inoculated with 1 ml of overnight culture. Cells were grown until absorbance at 600 nm reached ∼0.6, then the protein expression was induced with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside. After 4-h incubation at 37 °C, cells were harvested by centrifugation at 5000 × g for 10 min at 4 °C. Cells were resuspended in 50 ml of 50 mm Tris-HCl, pH 8.0, containing 50 mm NaCl, 2 mm EDTA, 0.4% (v/v) Triton X-100, and 0.4% (w/v) sodium deoxycholate. After the suspension was sonicated, the inclusion body pellet was obtained by centrifugation at 50,000 × g for 15 min at 4 °C. The pellet was resuspended in the same buffer and re-centrifuged, and the pellet was resuspended in 50 ml of 50 mm Tris-HCl, pH 8.0, containing 5 mm EDTA and 5 m urea. The pellet was stirred for 20 min at room temperature and then centrifuged at 100,000 ×g for 10 min at 4 °C. The washed inclusion body was resuspended in 10 ml of 50 mm Tris-HCl, pH 8.0, containing 8 m guanidinium chloride. Insoluble matter was removed by centrifugation at 100,000 × g for 10 min at 4 °C, and the supernatant was loaded onto a Sephadex G-25 column (2.5 × 25 cm) equilibrated with 50 mm Tris-HCl, pH 8.0, containing 5 m urea and 5 mm EDTA. The first major peak was collected (35 ml) and dialyzed against 50 mm Tris-HCl, pH 8.0, containing 1.5 m urea and then against 50 mm Tris-HCl, pH 8.0. The refolded C2 domain was purified using a Ni-NTA column (Qiagen) according to the manufacturer's instructions. Purity of all protein samples was higher than 90% electrophoretically. Aliquots of purified protein were stored at −20 °C. Activity of PLC was assayed by measuring the initial rate of Ins(4,5)P2 hydrolysis as described by Cifuentes et al. (23Cifuentes M.E. Honkanen L. Rebecchi M.J. J. Biol. Chem. 1993; 268: 11586-11593Abstract Full Text PDF PubMed Google Scholar) with some modifications. Small unilamellar vesicles (500 μm) containing 1% Ins(4,5)P2, a trace of [3H]Ins(4,5)P2 (2 × 104dpm) and bulk phospholipids (POPC, POPG, or POPS; each 495 μm) were prepared in 10 mm HEPES buffer, pH 7.0, containing 0.1 m KCl, 500 μg/ml BSA, and 0.5 mm Ca2+. Free calcium concentration was adjusted using a mixture of EGTA and CaCl2 according to the method of Bers (24Bers D.M. Am. J. Physiol. 1982; 242: C404-C408Crossref PubMed Google Scholar). The reaction was initiated by adding the indicated amount of enzyme, continued for 5 min, and quenched by adding 0.25 ml of 10% ice-cold trichloroacetic acid and 25 μl of 20% Trition X-100. Samples were kept on ice for 15 min, and the precipitate containing [3H]Ins(4,5)P2 was separated from the supernatant containing [3H]IP3 by centrifugation at 12,000 × g for 2 min at 4 °C. To the supernatant, 0.5 ml of CHCl3/MeOH (2:1), was added and the aqueous phase containing [3H]IP3 was extracted. Radioactivity of the hydrolyzed product was measured by liquid scintillation counting. Surface pressure (π) of solution in a circular Teflon trough was measured using a Wilhelmy plate attached to a computer-controlled tensiometer (25Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar). The trough (4 cm diameter × 1 cm depth) has a 0.5-cm deep well for a magnetic stir bar and a small hole drilled at an angle through the wall to allow an addition of protein solution. Five to ten milliliters of phospholipid solution in ethanol/hexane (1:9 (v/v)) was spread onto 10 ml of subphase (20 mm Tris-HCl, pH 7.5, containing 0.1m KCl and 0.5 mm free Ca2+) to form a monolayer with a given initial surface pressure (πo). The subphase was continuously stirred at 60 rpm with a magnetic stir bar. Once the surface pressure had been stabilized (after about 5 min), the protein solution (typically 50 μl) was injected into the subphase through the hole, and the change in surface pressure (Δπ) was measured as a function of time. Typically, the Δπ value reached a maximum after 20 min. The maximal Δπ depended on the protein concentration at the low concentration range and reached saturation when the protein concentration was higher than 3 μg/ml. Protein concentrations in the subphase were therefore maintained above such values to ensure the observed Δπ represented a maximal value. The preparation of vesicle-coated Pioneer L1 sensor chip (BIAcore) was described in detail elsewhere (26Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). The sensor surface was coated with POPC/POPS (7:3) or POPC/POPG (7:3) vesicles. In control experiments, the fluorescence intensity of the flow buffer after rinsing the sensor chip coated with vesicles incorporating 10 mm 5-carboxyfluorescien (Molecular Probes) was monitored to confirm that the vesicles remained intact on the chip. All experiments were performed with a control cell in which a second sensor surface was coated with POPC, because all C2 domains of PLC-δ isoforms showed negligible binding to POPC-coated chip. The drift in signal for both sample and control flow cells was allowed to stabilize below 0.3 resonance unit/min before any kinetic experiments were performed. All kinetic experiments were performed at 24 °C, and a flow rate of 60 μl/min in 10 mm HEPES, pH 7.4, containing 0.1 m NaCl and varying concentration of Ca2+. A high flow rate was used to circumvent mass transport effects. The association was monitored for 90 s and dissociation for 4 min. The immobilized vesicle surface was then regenerated for subsequent measurements using 10 μl of 50 mm NaOH. The regeneration solution was passed over the immobilized vesicle surface until the SPR signal reached the initial background value before protein injection. For data acquisition, five or more different concentrations (typically within a 10-fold range above or below the Kd) of each protein were used. After each set of measurements, the entire immobilized vesicles were removed by injection of 25 μl of 40 mm CHAPS, followed by 25 μl of octyl glucoside at 5 μl/min, and the sensor chip was re-coated with a fresh vesicle solution for the next set of measurements. All data were evaluated using BIAevaluation 3.0 software (BIAcore). For each trial, the signal was corrected against the control surface response to eliminate any refractive index changes due to buffer change. Furthermore, the derivative plot was used to monitor potential mass transport effects. Once these factors were checked for each set of data, the association and dissociation phases of all sensorgrams were globally fit to a 1:1 Langmuir binding model: [protein × vesicle] ↔ protein + vesicle. The association phase was analyzed using an equation,R=[kaC/(kaC+kd)]Rmax(1−e−(kaC+kd)(t−t0))+RIwhere RI = refractive index change,Rmax is the theoretical binding capacity,C is analyte concentration, ka is the association rate constant, and t0 is the time at start of fit data. The dissociation phase was analyzed using an equation, R=R0e−kd(t−t0) wherekd is the dissociation rate constant andR0 is the response at the start of fit data. The curve fitting efficiency was checked by residual plots and χ2. The dissociation constant (Kd) was then calculated from the equation, Kd =kd/ka. All constructs were ligated into the modified pIND vector (Invitrogen). An amino-terminal EGFP fusion was found to yield higher gene expression than the carboxyl-terminal counterpart. The spacer sequence between EGFP and the gene was AAA. A stable HEK293 cell line expressing the ecdysone receptor (Invitrogen) was used for all experiments. Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 and 98% humidity until 90% confluent. Cells were passaged into eight wells of a Lab-Tech-chambered coverglass for later transfection and visualization. Only cells between the 5th and 20th passages were used. 80–90% confluent cells in Lab-Tech-chambered cover glass wells were exposed to 150 μl of unsupplemented DMEM containing 0.5 μg of endotoxin-free DNA and 1 μl of LipofectAMINE reagent (Invitrogen) for 7–8 h at 37 °C. After exposure, the transfection medium was removed, and the cells were washed once with FBS-supplemented DMEM, and overlaid with FBS-supplemented DMEM containing Zeocin and 140 μg/ml ponasterone A to induce protein production. Images were obtained using a four-channel Zeiss 510 laser scanning confocal microscope. EGFP was excited using the 488-nm line of an argon/krypton laser. All experiments were carried out at the same laser power, which was found to induce minimal photobleaching over 30 scans, and at the same gain and offset settings on the photomultiplier tube. An LP 505 filter was used on channel 1 for all experiments. A ×63 magnification, 1.2 numerical aperture water immersion objective was used for all experiments. Cells for imaging were selected based on their initial intensity, which needed to fall in the upper third of the photomultiplier tube's range. The 510 imaging software provides an option for time series imaging and was used to control the time intervals for imaging. Ca2+-dependent translocation of C2 domains was monitored as follows: Thirty minutes before imaging, the cells were treated with 2 μl of Fura Red AM (Molecular Probes). Immediately before imaging, induction media were removed and the cells were washed with 150 μl of 2 mmEGTA and then overlaid with 150 μl of HEK buffer (1 mmHEPES, pH 7.4, containing 2.5 mm MgCl2, 1 mm NaCl, 0.6 mm KCl, 0.67 mm d-glucose, and 6.4 mm sucrose). After initially imaging a cell, 150 μl of HEK buffer containing ionomycin and various concentrations of Ca2+ was added to PLC-δ-C2-transfected cells. Control experiments were done with dimethyl sulfoxide in place of ionomycin. The electrostatic properties of the structure of PLC-δ1-C2 and homology models for PLC-δ3-C2 and PLC-δ4-C2 and mutants were calculated and visualized in the program GRASP (27Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5314) Google Scholar). In each panel of the figure, the red and blue meshes represent, respectively, the −25 and +25 mV electrostatic equipotential contours in 0.1 m KCl. All homology models were built based on the alignment of the sequence being modeled to the PLC-δ1-C2 sequence and using the structure of PLC-δ1-C2 as a template. Previous studies have suggested that PLC-δ1-C2 binds three Ca2+ ions (18Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar). Therefore, in the modeling studies, we used the structure of the PLC-δ1-C2 complexed with three lanthanum ions (1djg, residues 626–756) (18Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar), which we replaced with Ca2+ ions. Indistinguishable results were obtained with the structure of the C2 domain complexed with two calcium ions (1dji, residues 626–756) into which we modeled a third Ca2+ ion based on the structural alignment with the lanthanum-bound structure. Homology models were built for PLC-δ3-C2 and PLC-δ4-C2 as well as the S717D and K717E/R718E mutants of PLC-δ4-C2 with the program PRISM (28Yang A.S. Honig B. Proteins. 1999; 37: 66-72Crossref Scopus (51) Google Scholar). Hydrogen atoms were added to the heavy atoms of the structure and homology models with the program CHARMM (29Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comp. Chem. 1983; 4: 187-217Crossref Scopus (13874) Google Scholar). The structures with hydrogens were subjected to conjugate gradient minimization with a harmonic restraint force of 50 kcal/mole/Å2 applied to the heavy atoms located at the original crystallographic coordinates to minimize atomic clashes. Each model was evaluated using the program Verify 3D (30Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2539) Google Scholar), which scores structures according to how well each residue fits into its structural environment based on criteria derived from statistical analyses of the Protein Data Bank; all models scored well relative to the PLC-δ1-C2 domain structure. Rather than build separate models for both the Ca2+-free and Ca2+-bound forms of PLC-δ3-C2 and PLC-δ4-C2 and to facilitate comparison of their electrostatic properties, we assumed that the structures in the absence of Ca2+ are similar to those in its presence, and the calcium-free forms of the C2 domains were derived from the Ca2+-bound models by deleting the Ca2+ ions from the models' coordinate files. This assumption is consistent with the studies of PLC-δ1-C2 (18Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar). Confirming this, our results for Ca2+-free PLCδ1-C2 are insensitive to whether we used the Ca2+-free structure (1isd) or deleted the Ca2+ ions from the Ca2+-bound form. Four distinct PLC-δ isoforms have been identified so far (2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1212) Google Scholar). Although all four isoforms are homologous in general, some sequence variations are noticed in the calcium-binding loops in the C2 domains (see Fig.1). For instance, PLC-δ4-C2 has Ser in place of a calcium-ligating Asp residue (i.e.Asp708 for PLC-δ1). To determine how these variations affect the membrane binding properties of the C2 domains, we measured the membrane binding of the C2 domains of PLC-δ1, -3, and -4 isoforms by SPR analysis. We have shown that the SPR analysis allows direct determination of membrane association (ka) and dissociation (kd) rate constants for peripheral proteins (25Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar, 26Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). We first measured the binding of PLC-δ1-C2 to immobilized POPC/POPS (7:3) vesicles as a function of Ca2+concentration (see Table I). In the absence of Ca2+ (i.e. 0.1 mm EGTA), no appreciable binding was detected with protein concentration up to 1 μm, indicating that PLC-δ1-C2 has >μmaffinity for POPC/POPS (7:3) under this condition. As illustrated in Fig. 2, an increase in Ca2+ from 0.5 μm to 0.5 mmresulted in a 50-fold increase in the affinity (Kd) of PLC-δ1-C2 for POPC/POPS (7:3), demonstrating that it is a Ca2+-dependent membrane targeting domain. Interestingly, Ca2+ affected both ka(∼6-fold) and kd (∼9-fold) to a comparable degree. Our previous study indicated that nonspecific electrostatic interactions primarily accelerate the association of protein to anionic membrane surfaces, whereas hydrophobic interactions and specific interactions, whether electrostatic interactions or hydrogen bonding, mainly slow the membrane dissociation (26Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). Thus, it appears that Ca2+ ions are involved in both nonspecific electrostatic interactions and specific and/or hydrophobic interactions. As was the case with PLC-δ1-C2, PLC-δ3-C2 (up to 1 μm) showed no detectable affinity for immobilized POPC/POPS (7:3) vesicles in the absence of Ca2+. Again, the membrane affinity of PLC-δ3-C2 increased as a function of Ca2+. For this C2 domain, increasing the Ca2+ concentration from 0.5 μm to 0.5 mm led to a more pronounced