Phospholipase Cdelta 1 Is a Guanine Nucleotide Exchanging Factor for Transglutaminase II (Galpha h) and Promotes alpha 1B-Adrenoreceptor-mediated GTP Binding and Intracellular Calcium Release*

Kwang Jin BaekDagger §, Sung Koo KangDagger , Derek S. Damron, and Mie-Jae ImDagger ||

From the Dagger  Department of Molecular Cardiology and  Department of Anesthesiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, September 8, 2000, and in revised form, November 6, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Effectors involved in G protein-coupled receptor signaling modulate activity of GTPases through GTPase-activating protein or guanine nucleotide exchanging factor (GEF). Phospholipase Cdelta 1 (PLCdelta 1) is an effector in tissue transglutaminase (TGII)-mediated alpha 1B-adrenoreceptor (alpha 1BAR) signaling. We investigated whether PLCdelta 1 modulates TGII activity. PLCdelta 1 stimulated GDP release from TGII in a concentration-dependent manner, resulting in an increase in GTPgamma S binding to TGII. PLCdelta 1 also inhibited GTP hydrolysis by TGII that was independent from the alpha 1BAR. These results indicate that PLCdelta 1 is GEF for TGII and stabilizes the GTP·TGII complex. When GEF function of PLCdelta 1 was compared with that of the alpha 1BAR, the alpha 1BAR-mediated GTPgamma S binding to TGII was greater than PLCdelta 1-mediated binding and was accelerated in the presence of PLCdelta 1. Thus, the alpha 1BAR is the prime GEF for TGII, and GEF activity of PLCdelta 1 promotes coupling efficacy of this signaling system. Overexpression of TGII and its mutants with and without PLCdelta 1 resulted in an increase in alpha 1BAR-stimulated Ca2+ release from intracellular stores in a TGII-specific manner. We conclude that PLCdelta 1 assists the alpha 1BAR function through its GEF action and is primarily activated by the coupling of TGII to the cognate receptors.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Phospholipase C (PLC)1 delta 1 is a member of the PLC family that produces two second messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) (1). Among PLCs, PLCbeta isozymes are stimulated by guanine nucleotide-binding proteins (G protein) Gq and its family of proteins in response to activation of cell surface receptors. PLCgamma isozymes are activated by phosphorylation through growth factor receptors. A number of laboratories have reported that PLCdelta 1 is stimulated by a unique GTP-binding protein known as tissue transglutaminase (TGII, Galpha h) (2-5). TGII is a bifunctional enzyme, having GTPase and transglutaminase (TGase) activity (6, 7) and is present in the plasma membrane, cytosol, and nucleus in a variety of tissues and cells (6). Exchange of GDP to GTP by TGII is facilitated by activation of the cell surface receptors (4-9). These receptors include the alpha 1B-adrenoreceptor (AR) (5, 7, 8), alpha 1DAR (8), alpha -thromboxane receptor (9), and oxytocin receptor (4). The coupling of TGII with these receptors appears to be receptor subtype-specific (8, 9).

PLCdelta 1 is widely distributed and expressed highly in some tissues such as mouse heart (1, 10). Stimulation of the enzyme by TGII involving alpha 1BAR is modulated in a concerted fashion within the system. Thus, bimodal regulation of PLCdelta 1 activity has been observed depending on the Ca2+ levels and occupancy of guanine nucleotide by TGII (3, 11, 12). PLCdelta 1 is stimulated with low concentrations of Ca2+ by GTPgamma S·TGII (11). However, activity of the enzyme is subsequently inhibited by increasing Ca2+ concentrations where PLCdelta 1 is stimulated in the presence or absence of GDP. Similarly, Murthy et al. (3) have reported that GTP·TGII inhibits PLCdelta 1, while GDP·TGII stimulates the enzyme. The Ca2+ dependence is not clearly defined in this study. The TGII-mediated PLC stimulation is also modulated by the level of TGII expression (12). At low levels of TGII expression, the alpha 1BAR-stimulated PLC activity is increased, whereas the receptor-mediated PLC stimulation is inhibited when TGII is highly expressed. The PLCdelta 1 activity is also inhibited by IP3, competing with its substrate PIP2 for a binding site known as the pleckstrin homology domain (13-15). Studies have also demonstrated that an increase in the intracellular concentration of Ca2+ activates PLCdelta 1 (13, 16, 17), indicating that activation of PLCdelta 1 occurs secondarily in response to the receptor-mediated activation of other PLCs or Ca2+ channels. A GTPase activating-protein (GAP) for the small GTPase RhoA (RhoGAP) also activates PLCdelta 1 by direct association (18). All of these observations suggest that the PLCdelta 1 activity is regulated by multiple factors.

All known GTP binding subunits (Galpha ) of G proteins are GTPases, which hydrolyze GTP to GDP and orthophosphate (Pi). It is now recognized that a large number of regulators of G protein signaling (RGS) facilitate GTP hydrolysis by Galpha proteins (19-21). Independent from the actions of these RGS proteins, certain effectors in the G protein-coupled receptor system modulate GTP hydrolysis by Galpha proteins acting as GAP or guanine nucleotide exchanging factor (GEF) (22-26). For example, PLCbeta 1(22-24) and the gamma  subunit of cGMP phosphodiesterase (25) directly accelerates GTP hydrolysis by Galpha q and Galpha t, respectively. A recent study by Scholich et al. (26) has shown that adenylyl cyclase facilitates GTP binding to Gs, thereby functioning as both GEF and GAP. These findings indicate that the effector molecules modulate their cognate GTPases to terminate or facilitate the signals.

To date, none of the known heterotrimeric G proteins stimulates PLCdelta 1 (1, 27), and the mechanisms that regulate PLCdelta 1 activity remain complex and unclear. To further understand the characteristics and the interaction of PLCdelta 1 with TGII and its activation by TGII and the alpha 1BAR, we investigated the roles of PLCdelta 1 in the modulation of TGII activities, including the alpha 1BAR. Here, we report a distinct role of PLCdelta 1 in a coupling system involving the alpha 1BAR and TGII. PLCdelta 1 displays two regulatory functions for TGII. One is a GEF function, and the other is the inhibition of GTP hydrolysis by TGII. The GEF function of PLCdelta 1 promotes the alpha 1BAR-mediated GTP binding by TGII. Furthermore, our results also reveal that PLCdelta 1 is primarily activated by the activation of the alpha 1BAR through TGII, resulting in Ca2+ release from intracellular stores.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- Fura 2-AM was obtained from Texas Fluorescence Laboratories (Austin, TX), and G418 was obtained from Life Technologies, Inc. Radioactive materials including [alpha -32P]GTP (3000 Ci/mmol), [gamma -32P]GTP (3000 Ci/mmol), [35S]GTPgamma S (~1300 Ci/mmol), [3H]GDP (25-50 Ci/mmol), [3H]prazosin (79.8 Ci/mmol), and [3H]putrescine (37.5 Ci/mmol) were purchased from PerkinElmer Life Sciences. Heparin-agarose, wheat germ agglutinin-agarose, GTP-agarose, guinea pig liver TGase, hygromycin, and N,N'-dimethylcasein, phospholipids were purchased from Sigma. A monoclonal TGII antibody CUB7402 was obtained from NeoMarkers (Freemont, CA). A monoclonal PLCdelta 1 antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY), and a polyclonal Gq/11alpha antibody was from BIOMOL (Plymouth Meeting, PA). Fast Mono-Q Sepharose was obtained from Amersham Pharmacia Biotech. Nitrocellulose membrane BA85 was from Schleicher & Schuell. Norit A charcoal was from Serva (New York, NY).

Purification of Proteins-- PLCdelta 1 was expressed in DHalpha 5 cells and purified as described (2). The purity of the PLCdelta 1 preparation was >= 90%, as judged by silver staining, and neither GTPgamma S binding and TGase activity were observed. Guinea pig liver TGII was further purified using GTP-agarose as described (7, 28). The purity of the TGII preparation was >= 95% as judged by silver staining, and PLC activity was not found in the TGII preparation as determined by measurement of PIP2 hydrolysis (2). It should be noted that purified TGII was stable for <= 3-4 weeks in the presence of 10% glycerol at -80 °C. The alpha 1BAR was expressed in COS-1 cell (5) and partially purified in the presence of phentolamine by chromatography using heparin-agarose and wheat germ agglutinin-agarose as described (29). Contamination of TGII as well as other GTP-binding proteins was determined by measurement of TGase activity, direct photolabeling of GTPases with [alpha -32P]GTP (28), and immunoblotting with antibodies against TGII and Gq/11alpha (5). Galpha q (the virus was kindly provided by Dr. Elliott Ross at Northern Texas University, Dallas, TX) was expressed in Sf9 cells. Since we have found that Sf9 cells do not express TGII as judged by immunoblotting and measurement of TGase activity, Galpha q was partially purified by one-step chromatography using Mono Q-Sepharose. Membrane (5 mg/ml) prepared from Galpha q-expressed Sf9 cells was extracted with 1% sodium cholate in 50 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM 1,4-dithiothreitol, 3 mM EGTA, 1 mM EDTA, 5 mM MgCl2, 10 mM NaF, 30 µM AlCl3 as described (24, 27). The extract was diluted 10-fold with the same buffer containing 0.02% sucrose monolaurate (SM) and loaded onto Mono Q-Sepharose, which was pre-equilibrated with the same buffer containing 0.02% SM. The column was washed with the buffer and eluted with 300 mM NaCl and 10% glycerol in the same buffer. The amount of Galpha q was determined by immunoblotting with Gq/11alpha antibody. A single band with molecular mass of 42 kDa was detected with the antibody. Known concentrations of TGII were simultaneously immunoblotted to estimate the amounts of Galpha q. The protein preparations were aliquoted and stored at -80 °C until use. For each experiment, the protein preparations in the elution buffer was loaded onto a dried Sephadex G-25 column (3 ml) to remove the salts (29). The dried column was preequilibrated with an assay buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM 1,4-dithiothreitol, and 1.5 mM MgCl2). The recovery was ~40-50%, as determined by immunoblotting with the TGII and Gq/11alpha antibodies or TGase activity measurement for TGII. The recovery of the alpha 1BAR was ~50%, as determined by binding ability of [3H]prazosin (29). In addition, it should be noted that a nonhydrolyzable ATP analogue AppNHp (100 µM) was included in the assay buffer throughout the study, since it has been reported that TGII also binds ATP and hydrolyzes it (30).

Reconstitution-- Throughout the study, the proteins were reconstituted in phospholipid vesicles by a dilution method (31). Briefly, an appropriate amount of proteins was mixed with 0.2 mg of a clear phospholipid suspension (8 mg/ml in 0.2% SM solution) consisting of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (3:1:1, w/w/w). The concentrations of phospholipid mixture were 20-30 µg (final), and SM concentrations were ~0.008% (final). The reconstitution mixtures in the assay buffer were preincubated in an ice bath for 40 min throughout the study. For the studies involving alpha 1BAR (150 pM/tube), the samples were preincubated in the presence of 1 × 10-5 M (-)-epinephrine or 1 × 10-4 phentolamine.

Preparation of Radiolabeled Guanine Nucleotide-bound TGII-- A complex of [gamma -32P]GTP·TGII was prepared by incubating TGII (~50 nM) with 50 µM [gamma -32P]GTP (100,000 cpm/nM) in 300 µl of the assay buffer. After incubation at room temperature for 20 min, unbound [gamma -32P]GTP and [32P]Pi was removed by a dried Sephadex G-25 column which was preequilibrated with the assay buffer. The amounts of [gamma -32P]GTP·TGII complex were determined by a nitrocellulose membrane filter assay (29). Commercial [3H]GDP was lyophilized to remove ethanol and then reconstituted with water prior to use. The complex of [3H]GDP·TGII was prepared by incubating TGII (50 nM) with [3H]GDP (100 µCi) in 300-500 µl of the assay buffer at room temperature for 30 min. Unbound GDP was not removed since TGII has a low affinity for GDP (28).

Measurement of GTP Hydrolysis-- Since we have found that GTP hydrolysis by TGII is temperature-sensitive, the reaction was performed at room temperature. Single turnover GTP hydrolysis was determined with the [gamma -32P]GTP·TGII complex (~1 nM/tube) preparation. The complex was mixed with and without 4 nM PLCdelta 1 or with 4 nM heat-inactivated PLCdelta 1 (boiled for 20 min) in the assay buffer. At time 0, 100 µM cold GTP was added to prevent the rebinding of radiolabeled guanine nucleotide. At the indicated time, the samples were transferred to an ice-water bath, and the amount of [gamma -32P]GTP·TGII remaining was determined by the nitrocellulose filter method. A standard GTPase activity was also performed by charcoal absorption method (29). Briefly, vesicles containing proteins were mixed with 2 µM GTP plus 3 µCi of [gamma -32P]GTP in the assay buffer in a 100-µl final volume. The reaction was performed at room temperature for 20 min and stopped by addition of ice-cold Norit A charcoal suspension (5%, w/v, 900 µl) in 50 mM sodium phosphate buffer (pH 7.4). The reaction mixture was centrifuged for 20 min at 4 °C, and a 700-µl aliquot of the supernatants was withdrawn and recentrifuged under the same conditions. After a second centrifugation, the amount of [32P]Pi released in a 500-µl aliquot was determined by a beta -counter. To determine turnover, [35S]GTPgamma S binding was performed with the same samples at room temperature for 20 min.

Determination of [3H]GDP Release and GTPgamma S Binding-- Both experiments were carried out at 10 °C throughout the study. [3H]GDP·TGII (~1 nM) was incubated with 4 nM PLCdelta 1 or Ca2+-bound PLC-delta 1 or heat-inactivated PLCdelta 1. Ca2+-bound PLCdelta 1 was prepared by incubating of the enzyme with 30 µM Ca2+ at room temperature for 20 min. At this concentration of Ca2+, PLCdelta 1 was fully activated (2). The final concentration of Ca2+ was adjusted to 5 µM in the reaction mixtures. At time 0, 2 µM GTPgamma S was added to the reaction mixtures to prevent the rebinding of radiolabeled GDP. For the GTPgamma S binding experiments, the reaction was started by adding 2 µM [35S]GTPgamma S (1500 cpm/nM, final). Nonspecific binding was determined in the presence of 100 µM GTP or in the presence of 1 × 10-4 M phentolamine when the alpha 1BAR was reconstituted with TGII or Galpha q and PLCdelta 1. The time-dependent experiments were performed by transferring an aliquot (50 µl) to the ice-water bath at the indicated time points. The amounts of GTPgamma S binding by TGII or Galpha q and the remaining [3H]GDP·TGII complex were determined by the nitrocellulose filter method (29).

Measurement of TGase Activity-- After preincubation of TGII (4 nM) with various concentrations (0-10 nM) of PLCdelta 1, the TGase activity was measured in the presence of 1% N,N'-dimethylcasein, 1 µM putrescine (2.2 × 106 cpm/1 µM, final), and 300 µM Ca2+ at 25 °C for 20 min (28). The nonspecific TGase activity was determined in the presence of 1 mM EGTA.

Transfection and Cell Culture-- DNAs (10 µg/dish) of TGII and its mutants inserted into a neomycin-resistant selection vector pcDNA3.1-Neo (Invitrogene) were transfected to hamster leiomyosarcoma (DDT1-MF2) using LipofectAMINE method provided by the manufacturer (Life Technologies, Inc.). DDT1-MF2 cell has been shown to express the alpha 1BAR subtype only (32). TGII and its mutant expressed cells were selected using 500 µg/ml G418 in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum and 100 µg/ml penicillin, and 100 µg/ml streptomycin. After completion of the selection, PLCdelta 1 DNA (10 µg) inserted into a hygromycin selection vector pCEP4 (Invitrogene) was transfected into DDT1-MF2 cells expressing TGII and its mutants. The cells were selected with 500 µg/ml hygromycin in a growth medium containing 300 µg/ml G418. The established cells were maintained in the growth medium containing 300 µg/ml G418 and 300 µg/ml hygromycin.

Measurement of [Ca2+]i-- Cytosolic free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent Ca2+ indicator Fura 2-AM as described by Xu et al. (33). DDT1-MF2 cells (1 × 104 cells) were trypsinized and seeded on 35-mm glass culture dishes designed for fluorescence microscopy (Bioptech, Butler, PA). After the cells were incubated overnight in DMEM containing 10% heat-inactivated, the cells were further incubated in a serum-free DMEM for 24 h. The cells were then loaded with Fura 2-AM (2 µM) at room temperature for 40 min in Krebs Ringer (KR) buffer (25 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 11 mM glucose) containing 0.2% bovine serum albumin. After washing cells three times with KR buffer, the cells were kept in the tissue culture incubator until use. Before measurement of [Ca2+]i, the cells were washed three times with Ca2+-free KR buffer containing 1 × 10-6 M propranolol and 1 × 10-7 M rawalscine. The alpha 1BAR-mediated [Ca2+]i was determined by addition of 1 × 10-5 M (-)-epinephrine (final). Five cells, which were within the beam light, were selected to collect data. The reason for the selection of multiple cells is to minimize the variability of outcome, since expression level of proteins is expected to vary from a cell to a cell. The culture dishes were placed in a temperature-regulated chamber (37 °C). Fluorescence ratios were measured by an alternative wavelength time scanning method (dual excitation at 340 and 380 nm, emission at 500 nm). Estimation of [Ca2+]i were achieved by comparing the cellular ratio with fluorescence ratios acquired using Fura 2 (free acid) in buffer containing known concentrations of Ca2+. [Ca2+]i was calculated as described by Grynkiewicz et al. (34).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Effects of PLCdelta 1 on GTPase Function of TGII-- To assess whether PLCdelta 1 modulates GTPase activity of TGII, a single turnover hydrolysis of [gamma -32P]GTP by TGII was determined using a [gamma -32P]GTP·TGII preparation with and without PLCdelta 1 (Fig. 1A). In the absence of PLCdelta 1, the [gamma -32P]GTP hydrolysis by TGII was observed in a time-dependent manner and reached half-maximal GTP hydrolysis within 8 min. In contrast, when PLCdelta 1 was present, the [gamma -32P]GTP hydrolysis was extremely slow (~21% hydrolysis for 30-min incubation). The [gamma -32P]GTP hydrolysis was not inhibited by heat-inactivated PLCdelta 1, indicating that the inhibition is caused by the interaction of PLCdelta 1 with TGII. The PLCdelta 1-mediated inhibition of GTP hydrolysis by TGII was further examined using the charcoal absorption method. Equimolar (4 nM) of TGII and PLCdelta 1 or heat-inactivated PLC-delta 1 was mixed and incubated at room temperature for 20 min. The vesicles containing TGII alone or the heat-inactivated PLCdelta 1 produced [32P]Pi with a turnover of 1.2-1.5 mol-1 min-1. On the other hand, the vesicles containing TGII and PLCdelta 1 produced less [32P]Pi with a turnover of 0.24 mol-1 min-1. This demonstrates that PLCdelta 1 inhibits GTP hydrolysis by TGII, showing that PLCdelta 1 is not GAP for TGII.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Modulation of GTPase activity of TGII by PLCdelta 1. A, hydrolysis of [gamma -32P]GTP by TGII in the presence and absence of PLCdelta 1. [gamma -32P]GTP·TGII complex (~1 nM/tube) was mixed with and without PLCdelta 1 (4 nM/tube) as detailed under "Experimental Procedures." Boiled PLCdelta 1 was 4 nM. The reaction was performed at room temperature and stopped by transferring the samples in an ice-water bath. At time 0, amount of [gamma -32P]GTP·TGII was taken as 100%. B, PLCdelta 1 concentration-dependent GDP release from TGII. [3H]GDP·TGII (~1 nM) estimated by immunoblotting using TGII antibody was mixed with various concentrations of PLCdelta 1 (filled circle) or heat-inactivated PLCdelta 1 (open triangle). The GDP release was determined at 10 °C for 10 min. At time 0, amount of [3H]GDP·TGII without PLCdelta 1 was taken as 100%. C, time-dependent GDP release from TGII induced by PLCdelta 1. An equal amount of TGII (4 nM) and PLCdelta 1 (filled circle) or Ca2+-PLCdelta 1 (open square) was reconstituted. Filled triangle indicates [3H]GDP·TGII alone. The reactions were carried at 10 °C. Amount of [3H]GDP·TGII at time 0 was taken as 100%. The data present the means ± S.E. from one of the representative experiments in triplicate.

To determine whether PLCdelta 1 influences exchange of GDP to GTP by TGII, GDP release from TGII was determined (Fig. 1, B and C). To evaluate whether Ca2+-bound or unbound PLCdelta 1 exhibits this effect on TGII, PLCdelta 1 preincubated with Ca2+ (Ca2+-PLCdelta 1) was also tested. A [3H]GDP·TGII complex was reconstituted with various concentrations of PLCdelta 1. The results revealed that the GDP release from TGII was accelerated as a function of PLCdelta 1 concentration (Fig. 1B). The heat-inactivated PLCdelta 1 was unable to catalyze the [3H]GDP release, showing the specificity of PLCdelta 1 action on TGII. Furthermore, the [3H]GDP release from TGII induced by PLCdelta 1 was time-dependent, reaching half-maximal [3H]GDP release within 4 min (Fig. 1C). The samples containing [3H]GDP·TGII alone or [3H]GDP·TGII with Ca2+-PLCdelta 1 showed a slow [3H]GDP release with a similar rate. The result indicates that Ca2+-unbound PLCdelta 1 acts as GEF for TGII.

GEF action of PLCdelta 1 for TGII was further examined by determining GTPgamma S binding to TGII (Fig. 2, A and B). Consistent with the observations that PLCdelta 1 stimulated GDP release, the GTPgamma S binding of TGII was increased as a function of PLCdelta 1 concentration (Fig. 2A). At a 1:2 ratio of TGII versus PLCdelta 1, the GTPgamma S binding to TGII reached a plateau. PLCdelta 1 alone showed no GTPgamma S binding activity, indicating that the increased GTPgamma S binding is caused by the interaction of TGII with PLCdelta 1. When the GTPgamma S binding by TGII was determined as a function of incubation time with and without PLCdelta 1, the GTPgamma S binding in the presence of PLCdelta 1 was higher (~3-fold) than TGII alone and reached a maximum within 11 min (Fig. 2B). In the presence of Ca2+-PLCdelta 1, the GTPgamma S binding was similar to TGII alone, again demonstrating that Ca2+-PLCdelta 1 does not stimulate GTPgamma S binding to TGII. A slight increase of basal GTPgamma S binding by TGII was also observed in the presence of PLCdelta 1, probably due to interaction of the enzyme with TGII during preincubation.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of PLCdelta 1 on GTPgamma S binding by TGII. A, PLCdelta 1 concentration-dependent GTPgamma S binding by TGII. TGII (4 nM/tube) was mixed with various concentrations of PLCdelta 1. The reaction was carried out at 10 °C for 10 min. The nonspecific GTPgamma S binding was determined in the presence of 100 µM GTP. B, time-dependent GTPgamma S binding to TGII induced by PLCdelta 1. TGII (4 nM) were mixed with (filled circle) or without (filled triangle) PLCdelta 1 (8 nM) or with Ca2+-PLCdelta 1 (8 nM, open square). The reaction mixtures were incubated at 10 °C. C. Inhibition of TGase activity of TGII by PLCdelta 1. TGII was mixed with PLCdelta 1 (filled circle) or heat-inactivated PLCdelta 1 (open triangle). The TGase activity in the absence of PLCdelta 1 was taken as 100%. The data present the means ± S.E. from one of the representative experiments in triplicate.

It has also been shown that conformational changes in TGII modulate TGII activity, which are induced by the activators (35, 36). Thus, Ca2+-bound TGII can not function as GTPase, and GTP-bound TGII does not exhibit TGase activity. To determine whether PLCdelta 1 induces GTPase form of TGII, the TGase activity was determined in the presence of various concentrations of PLCdelta 1 or heat-inactivated PLCdelta 1 (Fig. 2C). The reaction was started with Ca2+ to prevent the activation of PLCdelta 1 and TGase of TGII. The results showed that Ca2+-mediated TGase activation was inhibited in a concentration-dependent manner by PLCdelta 1. Heat-inactivated PLCdelta 1 was unable to inhibit TGase activity, demonstrating that the inhibition of TGase activity is due to the interaction TGII with PLCdelta 1. In addition, to discern whether the cross-linking of proteins caused the decrease in the enzyme activity, samples treated under the same conditions were subjected to immunoblotting with TGII and PLCdelta 1 antibodies. Cross-linking of TGII-PLCdelta 1 or TGII·TGII or PLCdelta 1-PLCdelta 1 was not observed (data not shown). Taken together, these data clearly show that PLCdelta 1 is GEF for TGII and that the interaction of TGII with PLCdelta 1 induces a conformational change in TGII to become GTPase.

PLCdelta 1 Is a Helper of alpha 1BAR Function That Stabilizes the GTPase Conformation of TGII-- G protein-coupled receptors are GEFs for their cognate G proteins. Since the alpha 1BAR couples with both TGII and Galpha q (7, 8) but Galpha q does not stimulate PLCdelta 1 activity (27), we first evaluated the specificity of GEF function of PLCdelta 1 for TGII. The alpha 1BAR was reconstituted with either TGII or Galpha q in the presence and absence of PLCdelta 1, and GTPgamma S binding by TGII or Galpha q was determined (Fig. 3A). The results revealed that, although the alpha 1BAR was able to activate GTPgamma S binding to both TGII and Galpha q, GEF activity of PLCdelta 1 was specific for TGII. Thus, in the presence of PLCdelta 1, the alpha 1BAR-mediated GTPgamma S binding to TGII was further increased ~58%, whereas the level of the receptor-mediated GTPgamma S binding to Galpha q did not change. Moreover, PLCdelta 1 again stimulated GTPgamma S binding by TGII, but not by Galpha q. This observation is also consistent with the finding that PLCdelta 1 is not activated by Galpha q (27). Since GTP hydrolysis by TGII was inhibited in the presence of PLCdelta 1, we also determined whether the inhibition of GTP hydrolysis occurs in the presence of the alpha 1BAR (Fig. 3B). In the presence of PLCdelta 1, the rate of GTP hydrolysis was inhibited ~77% with the samples containing TGII and PLCdelta 1 and ~41% with the samples containing all three components. These results again indicate that PLCdelta 1 functions as a GTP hydrolysis-inhibiting factor (GHIF) and that there is a stable association of TGII with PLCdelta 1.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of PLCdelta 1 on the alpha 1BAR-mediated GTPgamma S binding to TGII. A, specificity of the GEF function of PLCdelta 1 for TGII. The indicated G proteins (4 nM/tube) were mixed with and without 4 nM PLCdelta 1 or with and without 150 pM alpha 1BAR. The reconstitution of proteins of interest was achieved in phospholipid vesicles. The GTPgamma S binding was determined in the presence of 2 µM [35S]GTPgamma S by incubating of the reaction mixtures at 10 °C for 10 min. B, effects of PLCdelta 1 on the alpha 1BAR-mediated GTP hydrolysis by TGII. The reconstituted samples indicated in the figure were incubated in the presence of 1 × 10-5 (-)-epinephrine or 20 µM GTPgamma S at room temperature for 20 min. Pi release was determined by charcoal absorption method as described under "Experimental Procedures." The [35S]GTPgamma S binding activity was also determined at room temperature for 20 min to calculate turnover of the GTPase activity. The data shown are the means ± S.E. from one of the representative experiments in triplicate.

To understand the mechanism of GEF activity of the alpha 1BAR versus PLCdelta 1 for TGII, the alpha 1BAR, TGII, and PLCdelta 1 were reconstituted, and the GTPgamma S binding activity of TGII was assessed under various conditions (Fig. 4). The alpha 1BAR-mediated GTPgamma S binding to TGII was evident, reaching a plateau within 6 min (Fig. 4A). When PLCdelta 1 was present, the receptor-mediated GTPgamma S binding was further increased (~47% at 2 min) and reached a plateau within 4 min. PLCdelta 1-mediated GTPgamma S binding to TGII was slow compared with alpha 1BAR-mediated GTPgamma S binding in both the presence and absence of PLCdelta 1. These data indicate that the alpha 1BAR is the prime GEF for TGII and that PLCdelta 1 functions secondarily. Although TGII alone showed no GTPgamma S binding at time zero, when the receptor and/or PLCdelta 1 were present, the basal level of GTPgamma S binding by TGII was increased. The order of the basal GTPgamma S binding was alpha 1BAR + TGII + PLCdelta 1 > alpha 1BAR + TGII > TGII + PLCdelta 1. To further understand the role of PLCdelta 1, the receptor and TGII were reconstituted with various concentrations of PLCdelta 1, and GTPgamma S binding by TGII was determined at 2 and 4 min (Fig. 4B). At the 2-min time point, GTPgamma S binding was increased as a function of PLCdelta 1 concentration. At the 4-min time point, GTPgamma S binding was reached maximum at 1:1 ratio of TGII and PLCdelta 1, and a further increase in the concentration of PLCdelta 1 did not increase the alpha 1BAR-mediated GTPgamma S binding, probably due to the limited amounts of TGII. When the TGII concentrations were varied at fixed amounts of PLCdelta 1, GTPgamma S binding by TGII was increased as a function of TGII concentration (Fig. 4C). Although maximum coupling efficacy was observed at 1:1 ratio of TGII and PLCdelta 1, a further increase of TGII concentration resulted in a decrease in the coupling efficacy. These results indicate that a level of each component governs the activation of GTP binding to TGII and that the alpha 1BAR and PLCdelta 1 induce GTPase conformation of TGII in a concerted way. The sequence of conformational changes of TGII would be: basal state of TGII, which can function as GTPase and TGase; the second state, GTPase conformation that is induced by the receptor and can reverse to the basal state; the third state, GTPase conformation induced by the receptor and stabilized by interacting with PLCdelta 1.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   GTPgamma S binding to TGII mediated by the alpha 1BAR in the presence and absence of PLCdelta 1. A, GTPgamma S binding to TGII in the presence and absence of the alpha 1BAR and/or PLCdelta 1. Indicated proteins (150 pM alpha 1BAR, 4 nM TGII, and PLC-delta 1) in the figure were reconstituted in phospholipid vesicles. At indicated time, an aliquot (50 µl) was transferred to a tube in an ice-water bath. Nonspecific GTPgamma S binding was determined in the presence 100 µM GTP or 1 × 10-4 M phentolamine. Standard error was 7-10% of the specific GTPgamma S binding. B, rate of the alpha 1BAR-mediated GTPgamma S binding to TGII in the presence of various concentrations of PLCdelta 1. The samples were incubated at 10 °C for 2 or 4 min. C, effects of TGII level on the coupling efficacy involving alpha 1BAR and PLCdelta 1. The samples were incubated at 10 °C for 4 min. All experiments were performed three times in duplicate, and the specific GTPgamma S binding is shown in the means ± S.E. from one of the representative experiments.

Overexpression of PLCdelta 1 Enhances [Ca2+] by Activation of the alpha 1BAR-- The role of PLCdelta 1 in facilitating coupling of the alpha 1BAR with TGII was further investigated using DDT1-MF2 cells stably expressing PLCdelta 1 without and with wild-type TGII (wtTG) and its mutants (Fig. 5). A TGII mutant (C-STG), which lacks TGase activity by mutation of Cys277 to Ser at TGase active site (37), was utilized to delineate GTPase versus TGase activity of TGII. Moreover, if PLCdelta 1 acts as a stabilizer of GTPase conformation of TGII through its GEF/GHIF activity, wtTG would provide the same result as C-STG does. To evaluate a specific interaction among alpha 1BAR, TGII, and PLCdelta 1, two TGII mutants were utilized; m3TG in which an alpha 1BAR interaction site on TGII was mutated (5), and Delta L656 (Delta delta 1TG) in which a PLCdelta 1 interaction site was deleted (38). Proteins were highly expressed, and the expression levels were comparable with each other (Fig. 5, A and B). It should be noted that a fast mobility of the m3TG on SDS-PAGE was also observed when the mutant was expressed in COS-1 cell (5). The reason is not clearly understood. However, differences in an apparent molecular weight were observed with TGIIs from different species, indicating that the mobility of TGII on SDS-PAGE gel is greatly affected by the primary structure of the enzyme (39). The slow mobility of Delta delta 1TG is expected, because of the deletion of 30 amino acid residues from C terminus (38). The coupling among alpha 1BAR, TGII, and PLCdelta 1 was assessed by measuring [Ca2+]i in a Ca2+-free buffer (Fig. 5C). The control cells (vector) transfected with vectors (pcDNA3.1 and pCEP4) displayed an increase in the level of [Ca2+]i in response to activation of the alpha 1BAR with (-)-epinephrine. The alpha 1-agonist-evoked peak increase in [Ca2+]i was further increased by ~59% when PLCdelta 1 (vector plus PLCdelta 1) was expressed, demonstrating that PLCdelta 1 increases the coupling efficacy of this signaling system. Since the experiments were performed in Ca2+-free buffer, the increase in [Ca2+]i is due to the release of Ca2+ from an intracellular store that is likely mediated by IP3 formed in response to PLCdelta 1 activation. Expression of wtTG or C-STG resulted in an increase in peak [Ca2+]i that was ~74% and ~63% greater than that observed in vector-transfected cells, respectively. The cells coexpressing wtTG and C-STG with PLCdelta 1 exhibited ~23% increase in peak [Ca2+]i compared with wtTG or C-STG alone. The reason for this limited increase of [Ca2+]i is probably due to a limited number of the alpha 1BAR, since the receptor is the prime GEF in this signaling system (see Fig. 4). The alpha 1-agonist-mediated Ca2+ release was due to the coupling of TGII with the alpha 1BAR and PLCdelta 1, because both m3TG- and Delta delta 1TG-expressing cells showed an increase in peak [Ca2+]i, which was ~53% less than the cells expressing wtTG or C-STG. Moreover, the peak [Ca2+]i was 20% less than the control cell (vector plus PLCdelta 1), and coexpression of PLCdelta 1 with these mutants did not significantly increase [Ca2+]i. Although a residual stimulation of Ca2+ release in activation of the alpha 1BAR is most likely due to the incomplete blocking of the interaction among these three proteins, it is also possible that the increase in [Ca2+]i in these cells is due to the coupling of the alpha 1BAR with other G proteins such as the Gq family of proteins. Preincubation of the cells with the alpha 1-antagonist prazosin or nonspecific PLC inhibitor U73122 completely abolished the alpha 1-agonist-mediated increase in [Ca2+]i (data not shown). To assess endoplasmic reticulum Ca2+ content, we treated the cells with thapsigargin (an inhibitor of endoplasmic reticulum Ca2+ pump, which stimulates Ca2+ release) at the end of the experiments (Fig. 5D). The thapsigargin-induced release of [Ca2+]i was the lowest in the cells coexpressing of PLCdelta 1 with wtTG or C-STG and correlated with the amounts of [Ca2+]i release induced by the activation of the alpha 1BAR with the cells expressing TGII and its mutants without or with PLCdelta 1. These data further demonstrate that the increase in [Ca2+]i is caused by release from the intracellular Ca2+ stores.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of PLCdelta 1 results in the increase in [Ca2+]i in response to the activation of the alpha 1BAR. A, expression level of TGIIs. Cell lysates (150 µg) were subjected to immunoblotting using a TGII antibody, followed by SDS-PAGE (10% gel). B, cell lysate (150 µg) was used to determine PLCdelta 1 by immunoblotting, followed by SDS-PAGE (10% gel). C, effects of PLCdelta 1 on the alpha 1BAR-mediated Ca2+ release in cells expressing PLCdelta 1 with and without wtTG and its mutants. D, thapsigargin-induced Ca2+ release. Concentration of thapsigargin was 5 µM, and level of [Ca2+] with the vector cells was taken as 100%. The data presented were obtained from the experiments shown in panel C.

To date, an effector protein acting as both GEF and GHIF for a GTPase has not been described in either a heterotrimeric or a monomeric GTPase signaling system. Our studies on the roles of PLCdelta 1 in regulation of TGII activities reveal that PLCdelta 1 exhibits GEF and GHIF activities for GTPase function of TGII. Evidence for the GEF function of PLCdelta 1 is that the enzyme facilitates GDP release from TGII and stimulation of GTPgamma S binding (Figs. 1 and 2). The inhibition of TGase activity by PLCdelta 1 and the GHIF activity of the PLCdelta 1 suggest that the interaction of PLCdelta 1 with TGII induces and stabilizes GTPase conformation of TGII. The GEF/GHIF activity of PLCdelta 1 displays independently from the alpha 1BAR. However, when the alpha 1BAR is present, the receptor is the prime GEF (Fig. 4). This conclusion is based on the observations that (i) the alpha 1BAR-mediated GTPgamma S binding is not additively enhanced in the presence of PLCdelta 1, (ii) PLCdelta 1 increases the rate of GTPgamma S binding mediated by the receptor, and (iii) PLCdelta 1-mediated GTPgamma S binding to TGII is slow as compared with that mediated by the alpha 1BAR.

The observation that overexpression of PLCdelta 1 results in elevation of the alpha 1BAR-mediated Ca2+ release from the intracellular Ca2+ stores is consistent with findings that PLCdelta 1 is the effector in TGII-mediated signaling pathway (2-5, 40). Furthermore, overexpression of wtTG and C-STG substantially enhances the alpha 1BAR-mediated Ca2+ release, and the TGII mutants m3TG and Delta delta 1TG greatly reduce the level of the alpha 1BAR-evoked Ca2+ release with or without overexpression of PLCdelta 1. Interestingly, the increase in [Ca2+]i was somewhat small when PLCdelta 1 was coexpressed with wtTG or C-STG (Fig. 5C). Although the reason for the limited Ca2+ release is probably due the limited number of cognate receptors, other mechanisms may be involved. Thus, the PLCdelta 1 activity is positively and negatively regulated by TGII depending on the Ca2+ level, expression level of TGII, and binding of guanine nucleotides (3, 11, 12). PLCdelta 1 can also be inhibited by its metabolite IP3 (13-15). Since coexpression of TGII with PLCdelta 1 increases basal IP3 formation (5), all of these factors would reduce the interaction capability of PLCdelta 1 with TGII. There were no significant differences in peak [Ca2+]i between wtTG- and C-STG-expressing cells, indicating that at the initiation of coupling of these three molecules, the increased Ca2+ level in cell does not affect the GTP binding by TGII. These results also support the findings that Ca2+-unbound PLCdelta 1 is GEF for TGII (Figs. 1C and 2B). Our results also indicate that PLCdelta 1 is catalytically activated by GTP·TGII, since the level of the endogenous PLCdelta 1 is sufficient to increase [Ca2+]i maximally when wtTG as well as C-STG was highly expressed (Fig. 5C).

Effectors such as PLCbeta 1 and cGMP phosphodiesterase as well as RGS proteins terminate GTPase function to prevent further activation of the effector themselves (19-26). In contrast, PLCdelta 1 facilitates the signaling involving the alpha 1BAR and TGII through its GEF/GHIF activity for TGII when the cognate receptors are present. This novel role of PLCdelta 1 is probably necessary to promote the production of second messengers and to overcome the nature of its regulation by multiple factors. The coupling efficacy of the alpha 1BAR with TGII in respect to the activation of the cognate PLC is poor as compared with that with Galpha q (7, 8, 12). In addition, two mechanisms for the regulation of PLCdelta 1 activity have been studied intensely: (i) a supporting role of other Ca2+-mobilizing receptor systems and (ii) an effector role in the receptor·TGII coupling system. Our results clearly support the latter mechanism; PLCdelta 1 is activated at the basal Ca2+ level in an intact cell by the alpha 1BAR through TGII. Operation of these two mechanisms probably depends on whether the cells express the cognate receptors and TGII. It has been reported that PLCdelta 1 is activated by the capacitative Ca2+ entry, following bradykinin stimulation in rat pheochromocytoma (PC-12) cell, which does not express TGII (17). In these regards, further study is required to establish physiological relevance.


    ACKNOWLEDGEMENTS

We thank Dr. Elliott Ross for kindly providing us with virus for Galpha q expression in Sf9 cells and valuable suggestions for the experiments. We also thank Dr. Jian-Fang Feng for purification of recombinant PLCdelta 1.


    FOOTNOTES

* This work was supported by Grant RO1-GM45985 from the National Institutes of Health.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.

§ Permanent address: Dept. of Biochemistry, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.

|| To whom correspondence should be addressed: Dept. of Molecular Cardiology (NB50), Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 444-216-8860; Fax: 444-216-9263; E-mail: imm@ccf.org.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008252200


    ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; G protein, guanine nucleotide-binding protein; GAP, GTPase-activating protein; GEF, guanine nucleotide exchanging factor; RGS, regulator of G protein signaling; GHIF, GTP hydrolysis-inhibiting factor (the terminology was used as to counterpart of GEF); GTPgamma S, guanosine 5-O-(3-thiotriphosphate); IP3, inositol 1,4,5-triphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; SM, sucrose monolaurate; PAGE, polyacrylamide gel electrophoresis; TGase, transglutaminase; TGII, tissue transglutaminase; AR, adrenoreceptor; AppNHp, adenyl-5'-yl imidodiphosphate; wtTG, wild-type tissue transglutaminase; C-STG, tissue transglutaminase mutant (Cys277 right-arrow Ser); DMEM, Dulbecco's modified Eagle's medium; KR, Krebs Ringer.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


1. Rhee, S. G., and Bae, Y. S. (1997) J. Biol. Chem. 272, 15045-15048[Free Full Text]
2. Feng, J.-F., Rhee, S. G., and Im, M.-J. (1996) J. Biol. Chem. 271, 16451-16454[Abstract/Free Full Text]
3. Murthy, S. N., Lomasney, J. W., Mak, E. C., and Lorand, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11815-11819[Abstract/Free Full Text]
4. Park, E. S., Won, J.-H., Han, K. J., Suh, P.-G., Ryu, S. H., Lee, H. S., Yun, H.-Y., Kwon, N. S., and Baek, K. J. (1998) Biochem. J. 331, 283-289[Medline] [Order article via Infotrieve]
5. Feng, J.-F., Gray, C. D., and Im, M.-J. (1999) Biochemistry 38, 2224-2232[CrossRef][Medline] [Order article via Infotrieve]
6. Im, M.-J., Russell, M. A., and Feng, J.-F. (1997) Cell. Signal. 9, 477-482[CrossRef][Medline] [Order article via Infotrieve]
7. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596[Medline] [Order article via Infotrieve]
8. Chen, S., Lin, F., Iismaa, S., Lee, K. N., Birckbichler, P. J., and Graham, R. M. (1996) J. Biol. Chem. 271, 32385-32391[Abstract/Free Full Text]
9. Vezza, R., Habib, A., and FitzGerald, G. A. (1999) J. Biol. Chem. 274, 12774-12779[Abstract/Free Full Text]
10. Small, K., Feng, J.-F., Lorenz, J., Donnelly, E. T., Yu, A., Im, M.-J., Dorn, G. W., II, and Liggett, S. B. (1999) J. Biol. Chem. 274, 21291-21296[Abstract/Free Full Text]
11. Das, T., Baek, K. J., Gray, C., and Im, M.-J. (1993) J. Biol. Chem. 268, 27398-27405[Abstract/Free Full Text]
12. Zhang, J., Tucholski, J., Lesort, M., Jope, R. S., and Johnson, G. V. (1999) Biochem. J. 343, 541-549[CrossRef][Medline] [Order article via Infotrieve]
13. Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10472-10476[Abstract]
14. Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S., and Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525[Abstract/Free Full Text]
15. Cifuentes, M. E., Delaney, T., and Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948[Abstract/Free Full Text]
16. Allen, V., Swigart, P., Cheung, R., Cockroft, S., and Katan, M. (1997) Biochem. J. 327, 545-552[Medline] [Order article via Infotrieve]
17. Kim, Y.-H., Park, T.-J., Lee, Y. H., Baek, K. J., Suh, P.-G., Ryu, S. H., and Kim, K.-T. (1999) J. Biol. Chem. 274, 26127-26134[Abstract/Free Full Text]
18. Homma, Y., and Emori, Y. (1995) EMBO J. 14, 286-291[Abstract]
19. Berman, D. M., and Gilman, A. G. (1998) J. Biol. Chem. 273, 1269-1272[Free Full Text]
20. De Vries, L., and Gist Farquhar, M. (1999) Trends Cell Biol. 9, 138-144[CrossRef][Medline] [Order article via Infotrieve]
21. Hepler, J. R. (1999) Trends Pharmacol. Sci. 20, 376-382[CrossRef][Medline] [Order article via Infotrieve]
22. Berstein, G., Blank, J. L., Jhon, D. Y., Exton, J. H., Rhee, S. G., and Ross, E. M. (1992) Cell 70, 411-418[Medline] [Order article via Infotrieve]
23. Biddlecome, G. H., Berstein, G., and Ross, E. M. (1996) J. Biol. Chem. 271, 7999-8007[Abstract/Free Full Text]
24. Chidiac, P., and Ross, E. M. (1999) J. Biol. Chem. 274, 19639-19643[Abstract/Free Full Text]
25. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417[CrossRef][Medline] [Order article via Infotrieve]
26. Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331[Abstract/Free Full Text]
27. Hepler, J. R., Kozasa, T., Smrcka, A. V., Simon, M. I., Rhee, S. G., Sternweis, P. C., and Gilman, A. G. (1993) J. Biol. Chem. 268, 14367-14375[Abstract/Free Full Text]
28. Feng, J.-F., Readon, M., Yadav, S. P., and Im, M.-J. (1999) Biochemistry 38, 10743-10749[CrossRef][Medline] [Order article via Infotrieve]
29. Im, M.-J., Riek, R. P., and Graham, R. M.. (1990) J. Biol. Chem. 265, 18952-18960[Abstract/Free Full Text]
30. Iismaa, S. E., Chung, L., Wu, M. J., Teller, D. C., Yee, V. C., and Graham, R. M. (1997) Biochemistry 36, 11655-11664[CrossRef][Medline] [Order article via Infotrieve]
31. Im, M.-J., Gray, C., and Rim, A. J. (1992) J. Biol. Chem. 267, 8887-8894[Abstract/Free Full Text]
32. Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefknowitz, R. J., Caron, M. G., and Kobilika, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7159-7163[Abstract]
33. Xu, Y., Zhu, K., Hong, G., Wu, W., Baudhuin, L. M., Xiao, Y., and Damron, D. S. (2000) Nat. Cell Biol. 2, 261-267[CrossRef][Medline] [Order article via Infotrieve]
34. Grynkiewicz, G., Poenie, M., and Ysien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract]
35. Monsonego, A., Friedmann, I., Shani, Y., Eisenstein, M., and Schwartz, M. (1998) J. Mol. Biol. 282, 713-720[CrossRef][Medline] [Order article via Infotrieve]
36. Achyuthan, K. E., and Greenberg, C. S. (1987) J. Biol. Chem. 262, 1901-1906[Abstract/Free Full Text]
37. Lee, K. N., Arnold, S. A., Birckbichler, P. J., Patterson Jr, M. K., Fraij, B. M., Takekeuchi, Y., and Carter, H. A. (1993) Biochim. Biophys. Acta 1202, 1-6[Medline] [Order article via Infotrieve]
38. Hwang, K. C., Gray, C. D., Sivasubramanian, N., and Im, M.-J. (1995) J. Biol. Chem. 270, 27058-27062[Abstract/Free Full Text]
39. Baek, K. J., Das, T., Gray, C., Antar, S., Murugesan, G., and Im, M.-J. (1993) J. Biol. Chem. 268, 27390-27397[Abstract/Free Full Text]
40. Wu, J., Liu, S.-L., Zhu, J.-L., Norton, P. A., Nojiri, S., Hoek, J. B., and Zern, M. A. (2000) J. Biol. Chem. 275, 22213-22219[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.