Evidence that phospholipase delta1 is the effector in the Gh (transglutaminase II)-mediated signaling.

A new class of GTP-binding protein transglutaminase II (Gh) couples to a 69-kDa phospholipase C (PLC). An 8-amino acid region (Leu665-Lys672) of the α-subunit of Gh (Gαh) is involved in interaction and activation of PLC, an observation that has now been used to characterize the 69-kDa PLC further. A 20-amino acid peptide corresponding to Leu654-Leu673 of Gαh was used to prepare an affinity resin. On incubation with a partially purified PLC preparation from rat liver membranes, the affinity resin-bound ∼69- and 85-kDa proteins were recognized by an antibody to the 69-kDa PLC. Both purified 69-kDa PLC and PLC-δ1 bound to the affinity resin; moreover, antibodies to PLC-δ1 recognized the 69-kDa PLC, and antibodies to the 69-kDa PLC recognized PLC-δ1. A synthetic peptide corresponding to Leu661-Lys672 of Gαh inhibited the binding of PLC-δ1 to the affinity resin and also stimulated PLC-δ1. Reconstitution of PLC-δ1 with GTPγS (guanosine 5′-3-O-(thio)triphosphate)-activated Gh resulted in activation of PLC-δ1. Antibodies to Gαh also coimmunoprecipitated PLC-δ1 upon activation of Gh. These findings indicate that PLC-δ1 is the effector of Gh-mediated signaling.

Purification of Other Proteins-Crude PLC was prepared by one step chromatography using heparin-agarose (see Ref. 15). Rat liver membranes (1 g of protein) were extracted with 0.4% SM and 200 mM NaCl in 20 mM Hepes (pH 7.4) containing 5 mM EDTA, 5 mM EGTA, 1 mM DTT, and the protease inhibitors listed above. The extract was diluted 3 times with the same buffer and loaded onto a heparin-agarose column (20 ml). The column was washed with the same buffer containing 200 mM NaCl and 0.02% SM. Bound PLC was eluted using a linear gradient of NaCl (0.2-1.2 M) in the above buffer containing 0.02% SM. Fractions containing PLC activity were pooled and used for the study. The 69-kDa PLC was purified according to the method of Das et al. (15), and G h was purified according to the method of Im et al. (19).
Preparation of Peptide-Sepharose-Peptide cross-linked to Sepharose was synthesized using CNBr-Sepharose 4B with a slight modification of the method recommended by Pharmacia. The peptides (P5 or P6, shown in Fig. 1) were dissolved in minimum amounts of dimethyl sulfoxide (Me 2 SO) and diluted with equal amounts of water. After washing with 10 mM HCl, the activated resin (10 ml, wet volume) was suspended in a 100 mM NaHCO 3 /Me 2 SO (1:1, v/v) solution (40 ml), and the peptide (20 -25 mM) diluted in the NaHCO 3 /Me 2 SO solution was added. After incubation at room temperature for 1 h, the resin was washed with the NaHCO 3 /Me 2 SO solution. The resin in the NaHCO 3 / Me 2 SO solution was further incubated with 100 mM ethanolamine under the same condition described above. The resin was washed with 20 mM Tris⅐HCl (pH 7.4) containing 100 mM NaCl. The peptide-Sepharose was stored in the same buffer containing 0.02% azide at 4°C.
Binding of PLCs to Peptide-Sepharose-For the binding of PLCs to peptide-Sepharose, PLC preparations (pure, 20 ng ϩ 1 g of bovine serum albumin; crude, 50 g) were incubated with the resins (30 l of gel/tube) at 4°C for 1 h with gentle rotation. The final volume was 100 l in HSD buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, and 0.5 mM DTT) containing 0.01% SM. After centrifugation at 1,900 ϫ g for 5 min, supernatants and pellet fractions were collected, and the pellets were washed three times with 1 ml HSD buffer containing 0.05% SM. PLC activity was measured in both supernatant and pellets at 30°C for 10 min according to the method of Im et al. (20). For the immunoblotting, peptide affinity resins (0.5 ml) were washed with water and then equilibrated with HSD buffer containing 0.01% SM. The resins were incubated with crude PLC preparation (200 g) at 4°C for 1 h with gentle rotation. The resins were transferred to small columns (3 ml) and then washed with 10 ml of HSD buffer containing 0.05% SM. The bound proteins were eluted in a small volume (300 -500 l) with a 9 M urea, 1 M NaCl solution.
Reconstitution of Peptides and G h with PLC-␦1-The purified PLC-␦1 was reconstituted with peptides in detergent solution and with G h in phospholipid vesicles (15). PLC-␦1 (7 ng/tube) in HSD buffer containing 0.02% SM was incubated with various concentrations of peptides (0 -220 M). The PLC-␦1 activity was measured in the presence of 3.74 nM of free Ca 2ϩ at 30°C for 8 min. The free Ca 2ϩ concentration was obtained in the presence of 0.8 mM EGTA and 75 M CaCl 2 in HSD buffer. Reconstitution of G h with PLC-␦1 was achieved by the dilution method (20). G h (6 pmol) was preincubated with GTP␥S, GDP, or buffer only in the presence of 5 mM MgCl 2 at 30°C for 30 min. Ligandpretreated G h was mixed with PLC-␦1 (200 ng) and a phospholipid mixture (phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine; 3:1:1) in an ice bath. The PLC activity was measured with various concentrations of CaCl 2 (0 -16 M) at 30°C for 8 min (15,20).

FIG. 2.
Binding of PLC to P5 and P6 affinity resins. A, a partially purified PLC preparation (50 g) was incubated at 4°C for 1 h with P5 or P6 peptide affinity resins (30 l of resin) or ethanolamine crosslinked to Sepharose 4B (Resin), that had been equilibrated with HSD buffer containing 0.01% SM. The activity of resin-bound PLC was determined by incubating the samples at 30°C for 10 min as detailed under "Experimental Procedures." Data are means of three independent experiments, each performed in duplicate. B, the partially purified PLC preparation (200 g) was applied to P5 or P6 affinity columns (0.5 ml), which were then washed with HSD buffer containing 0.01% SM. The bound proteins were eluted in three portions (300 -500 l) with a 1 M NaCl solution containing 9 M urea, concentrated with an Amicon Centricon-30 filtration cell to 100 l, and subjected to immunoblot analysis using a polyclonal antibody to the 69-kDa PLC. PLC lane contains purified 69-kDa PLC. Arrows indicate immunoreactive protein, and the positions of molecular size standards (in kilodaltons) are also indicated on the right. A, the P6 affinity resin (30 l) was incubated with 20 ng of either 69-kDa PLC or PLC-␦1 at 4°C for 1 h. Bovine serum albumin (1 g) was included in the incubation mixture to prevent nonspecific binding. The mixture was then centrifuged at 1,900 ϫ g for 5 min, the pellets and supernatants were collected, and the pellets were washed three times with 1 ml of HSD buffer containing 0.05% SM. The pellets and supernatants were assayed for PLC activity in a final volume of 100 l. Data shown are the means of two independent experiments, each performed in duplicate. B, immunoblot analysis of purified 69-kDa PLC and PLC-␦1 (1 g each) with a monoclonal antibody to PLC-␦1.
The PIP 2 concentration was 100 M (1200 cpm/nmol) in a 100-l final volume.
Western Blotting-Polyclonal antibody against 69-kDa PLC was generated in New Zealand White rabbits according to the method of Baek et al. (21). 69-kDa PLC (40 -60 g in 200 l/rabbit) was used to raise the antibody. Rabbit antisera were characterized by immunoblots with purified 69-kDa PLC (21,22), except antibody cross-reactivity was determined using chemiluminescence. Anti-rabbit Ig horseradish peroxidase (1:1000 dilution) was used for the secondary antibody. Immunoblotting with monoclonal antibody against bovine brain PLC-␦1, PLC-␤1 and PLC-␥1 was performed using the same protocol (21,22). Anti-mouse Ig horseradish peroxidase was used for the secondary antibody.
Coimmunoprecipitation-For the coimmunoprecipitation studies, G␣ h (monoclonal antibody raised against guinea pig liver transglutaminase and G␣ h7 ) antibodies and preimmune sera were cross-linked with protein A-agarose (15,23). Reconstituted samples of G h with PLC-␦1 in HSD buffer containing 0.02% SM were incubated with or without 2 M GTP␥S in the presence of 2 mM MgCl 2 . After incubation at room temperature for 30 min, the samples were further incubated with the antibody affinity-agarose (30 l suspension/tube in HSD buffer) at 4°C for 2 h with gentle rotation. The supernatant and pellets were collected by centrifugation at 1,900 ϫ g for 5 min. The pellets were washed three times with 1 ml HSD buffer containing 0.05% SM and 2 mM MgCl 2 . PLC activity was measured in both supernatant and pellets at 30°C for 10 min (15).
Other Assays-G h concentration was measured using 1 M [ 35 S]GTP␥S (specific activity, 1000 -1500 cpm/pmol) (19). The protein concentration was determined by the method of Bradford (24) using a Bio-Rad protein determination kit and bovine serum albumin as a standard.

RESULTS AND DISCUSSION
We have shown previously that a stretch of 12 amino acids (P4, Leu 661 -Lys 672 ) near the COOH terminus of G␣ h contained the high affinity PLC binding site (16) (Fig. 1). This observation was further applied to identify the PLC isozyme activated by G␣ h . We synthesized four peptides (P3-P6) (Fig. 1) and crosslinked P5 and P6 to CNBr-Sepharose 4B for use as peptideaffinity resins. A partially purified PLC preparation from rat liver membranes was incubated with the peptide affinity resins and their ability to bind PLC was determined ( Fig. 2A). The P6 affinity resin bound 3-6 times more PLC activity than the P5 affinity resin or resin alone did, consistent with P6 containing the PLC binding site. The partially purified PLC preparation was then loaded onto a P6-Sepharose column, and the bound proteins were eluted and subjected to immunoblot analysis with antibodies to the 69-kDa PLC (Fig. 2B). The antibodies recognized two proteins with molecular sizes of ϳ69 and 85 kDa (lane 2) as well as the purified 69-kDa PLC (lane 3). The antibodies did not recognize any proteins in the eluate from the P5 affinity resin (lane 1). Furthermore, nonimmune serum did not recognize the ϳ69and 85-kDa proteins in the eluate from the P6 affinity column (data not shown). These results suggested that the 85-kDa protein, which has approximately the same molecular size as PLC-␦1, is structurally similar to the 69-kDa PLC. PLC activity measurements revealed that both purified 69-kDa PLC and PLC-␦1 bound to the P6 affinity resin (Fig. 3A). Furthermore, both purified proteins were recognized by a monoclonal antibody to PLC-␦1 (Fig. 3B), suggesting that the 69-kDa PLC is structurally similar to PLC-␦1 and may be a proteolytic fragment of this enzyme. The polyclonal antibodies to the 69-kDa PLC cross-reacted with PLC-␦1 (data not shown). The 69-and 85-kDa proteins present in the partially purified preparations that bound to the P6 affinity resin were recognized by the monoclonal antibody to PLC-␦1 but not by monoclonal antibodies to PLC-␤1 or PLC-␥1.
The specificity of PLC-␦1 binding to the P6 affinity resin was then evaluated. PLC-␦1 was incubated with P5 or P6 affinity resins in the presence or absence of P4 (Fig. 4A). PLC-␦1 did bind to the P6 affinity resin but not to the P5 affinity resin, and the peptide P4 blocked the PLC-␦1 binding to the P6 resin. In addition, peptide P3 did not inhibit the binding of PLC-␦1 to the P6 affinity resin, showing that the P4 region contributes to the binding of PLC to G␣ h (data not shown). Since this P4 region of G␣ h interacts with and activates PLC (16), we investigated whether peptide P4 activates PLC-␦1. Indeed, the peptide P4 did activate PLC-␦1 in a concentration-dependent manner with an EC 50 of ϳ50 M, and a maximal activation was reached at Ն150 M (Fig. 4B). Peptide P3 did not stimulate PLC-␦1 (Fig.  4B), indicating that stimulation of PLC-␦1 by P4 is specific.
Finally, coupling between PLC-␦1 and G h reconstituted in phospholipid vesicles was investigated. We have previously reported that EGTA inhibits the coupling of G h with 69-kDa PLC (15). Similarly, coupling between G h and PLC-␦1 was not observed in the presence of chelator. Therefore, coupling was assessed in the absence of chelator under the conditions de- scribed previously (15,20). Reconstitution of PLC-␦1 with G h reduced basal PLC activity by two-thirds (data not shown). Activated (GTP␥S-bound) G h increased PLC-␦1 activity in a Ca 2ϩ concentration-dependent manner (Fig. 5A); the activity was maximal at Յ 12 M Ca 2ϩ , similar to the concentration required for maximal activation of the 69-kDa PLC by G h (15). Enzyme activity was less sensitive to Ca 2ϩ in the presence of GDP or buffer alone; under these conditions, PLC-␦1 was not fully activated at Ն16 M Ca 2ϩ . Thus, activation of PLC-␦1 by G h resulted in an increase in the affinity of the enzyme for Ca 2ϩ , as observed when G h and the 69-kDa PLC were coupled (15). We also showed that antibodies to G␣ h coimmunoprecipitated PLC-␦1 when the enzyme was reconstituted with GTP␥Sbound G h (Fig. 5B); PLC-␦1 was not coimmunoprecipitated in the absence of GTP␥S. Antibodies from nonimmune serum coupled to protein A-agarose did not coprecipitate PLC-␦1 in the presence or absence of GTP␥S (data not shown).
It has been suggested that low molecular mass PLCs might be proteolytic fragments of PLC-␤, ␥, and ␦ isozymes (2,25). Previous studies have indicated that the 69-kDa PLC is probably a proteolytic fragment of PLC-␦1. Thus, the elution profile of 69-kDa PLC is similar to PLC-␦1 on ion exchange chromatographic resins, particularly on heparin-agarose (15). Digestion of PLC-␦1 by proteases yields fragments of 52-68 kDa (25); in the presence of PIP 2 , trypsin generates a 68-kDa fragment from PLC-␦1 (26). Indeed, the original purification of the 69-kDa PLC-G h complex may have contributed to an increased sensitivity of PLC-␦1 to proteases, because the complex was induced by ␣ 1 -adrenoreceptor activation in the presence of GTP (15), which increases the binding of PIP 2 to PLC-␦1. On the other hand, we previously stated that the purified 69-kDa PLC did not cross-react with antibodies to PLC-␦1 and that G h did not stimulate PLC-␦1 activity (15). The reason for the failure to stimulate PLC-␦1 or to detect the 69-kDa PLC by PLC-␦1 antibody is not clearly understood. It is probably due to the viability of the purified enzymes and affinity of the antibody for the 69-kDa PLC, since we previously used a polyclonal antibody to PLC-␦1. For the previous reconstitution studies, PLC-␦1 was purified from the cytosolic fraction of bovine brain. Although it is not known whether cytosolic PLC-␦1 is functionally different from membrane associated enzyme, the source of the enzyme could be another factor. Nonetheless, our present studies clearly indicate that 69-kDa PLC is a proteolytic fragment of PLC-␦1 and that PLC-␦1 is the effector of G h signaling. B, coimmunoprecipitation of PLC-␦1 by antibodies to G␣ h that were coupled to protein A-agarose. PLC-␦1 (50 ng) was reconstituted with G h (2 pmol) in HSD buffer containing 0.02% SM. Immunoprecipitation was then performed with antibodies to G␣ h as described under "Experimental Procedures," and PLC activity in the precipitates and supernatants was assayed. PLC activity in the original samples was taken as 100%. Data in A and B are means of three independent experiments, each performed in duplicate.