©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction Site of GTP Binding G (Transglutaminase II) with Phospholipase C (*)

(Received for publication, August 14, 1995; and in revised form, September 12, 1995)

Ki-Chul Hwang Caroline D. Gray Natarajan Sivasubramanian Mie-Jae Im (§)

From the Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The GTP binding Galpha(h) (transglutaminase II) mediates the alpha-adrenoreceptor signal to a 69-kDa phospholipase C (PLC). Thus, Galpha(h) possesses both GTPase and transglutaminase activities with a signal transfer role. The recognition sites of this unique GTP binding protein for either the receptor or the effector are completely unknown. A site on human heart Galpha(h) (hhGalpha(h)) has been identified that interacts with and stimulates PLC. Expressed mutants of hhGalpha(h) with deleted C-terminal regions lost the response to(-)-epinephrine and GTP and failed to coimmunoprecipitate PLC by the specific G antibody. The interaction regions were further defined by studies with synthetic peptides of hhGalpha(h) and a chimera in which residues Val-Lys of hhGalpha(h) were substituted with Ile-Ser residues of human coagulation factor XIIIa. Thus, eight amino acid residues near the C terminus of hhGalpha(h) are critical for recognition and stimulation of PLC.


INTRODUCTION

The Galpha(h) protein, transglutaminase II (TGase II), (^1)is unique in that the enzyme exhibits two distinct enzyme activities, namely guanosine triphosphatase (GTPase) and TGase, with a signal transfer role ((1) ; see also (2) and (3) ). The GTPase function of Galpha(h) differs from other TGases, coagulation factor XIIIa (FXIIIa), keratinocyte, and epidermal transglutaminases (4) . Galpha(h), which is species specific in molecular mass, directly interacts with alpha(1)-adrenoreceptor (5, 6) and a 69-kDa PLC in the activation process(7, 8) . Physiological TGase role of Galpha(h) remains unclear(4) . However, it has been suggested that TGase II is involved in control of cell growth and differentiation (9, 10, 11) and activation of cytosolic phospholipase A(2)(12) .

The amino acid sequences of all TGases including Galpha(h) show high homology in the middle portions of the polypeptides, which include the TGase active site and a calcium binding region(13) . However, the N- and C-terminal regions of Galpha(h) do not share sequence homology among TGases. This divergence is particularly greater at the C-terminal domain of Galpha(h), giving rise to the hypothesis that this region may play a significant role in hormone signaling. In this study, evidence for a direct interaction between the region of Galpha(h) and PLC is demonstrated. This interaction activates PLC.


EXPERIMENTAL PROCEDURES

Isolation and Mutagenesis of hhGalpha cDNA

Full-length cDNA of human heart Galpha(h) (hhGalpha(h)) was isolated from a human heart cDNA library by polymerase chain reaction (PCR) using two oligonucleotides, CCCGACCATGGCCGACGAGGAGCTGGTCT (5`-primer) and TGGGCCAGGGGCACATTCCATTTC (3`-primer), synthesized from the known nucleotide sequences of human endothelial TGase II(14) . After the PCR product (2.1 kilobases) was cloned into the pCRII without purification, the insert was digested with KpnI and NotI and cloned into the modified eukaryotic expression vector pMT2` to yield pMT2`hhGalpha(h)(1) . Four 10-amino acid-truncated mutants of hhGalpha(h) were generated by introducing a TAA stop codon using the following oligonuceotides: 1) CTTCACAGCCTTCAGCTTGTCGCT, 2) GTTCACCACCAGCTTGTGGAGGCC, 3) GAGCGGCACGAGGTCCATTCTCAC, and 4) TTCCTCCCCTGCCTCCACGGGGTC. The hhGalpha(h)/human FXIIIa chimera was constructed by ligation of PCR products generated from two different sets of primers containing nucleotide sequences of human FXIIIa (5`-primer, 5`-GAATTCGAATTCCCCGACCATGGCCGAGGAGCTGGTCTTA-3` and 3`-primer, 5`-GCTGGCTATCAGCTTGTGGAGGCCCATGTGGAGCGGCACGAGGTC-3`; 5`-primer, 5`-ATGAGCAGTGACTCCAAGGCTGTGAAGGGCTTCCGGAATGTCATC-3` and 3`-primer, 5`- GCGGCCGCGCGGCCGCTGGGCCAGGGGCACATTCCATTTC-3`) (single underline denotes nucleotide sequence from human FXIIIa; double underline denotes 5`-primer encoding two EcoRI restriction sites at 5`-primer and two NotI restriction sites at 3`-primer). Each PCR product was treated with Klenow fragment of Escherichia coli DNA polymerase I and digested with EcoRI or NotI. The blunt ends of two fragments were ligated and cloned into the eukaryotic expression vector pMT2`. Orientation of the constructs was confirmed by restriction enzyme mapping and DNA sequencing.

Expression of hhGalpha Proteins and Preparation of Membranes

Transfection and membrane preparation were performed using the method of Nakaoka et al.(1) . COS-1 cells (5 times 10^6 per 100-mm dish) were cotransfected with plasmids containing alpha-adrenoreceptor cDNA (4-5 µg of cDNA) and hhGalpha(h) or its mutants (8-10 µg of cDNA) using the DEAE-dextran method. The cells were grown for 48-72 h after transfection. The membranes prepared from the transfected COS-1 cells were suspended in a buffer (20 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol, and protease inhibitors) and were stored at -80 °C until use.

Analysis of Expressed hhGalpha Protein and Its Mutants

All expressed proteins were estimated by immunoblotting with guinea pig liver TGase II and G antibodies(1, 6) . Membrane proteins (100 µg) were solubilized with 1% sodium cholate and subjected to SDS-polyacrylamide gel electrophoresis (10% gel). The proteins were transferred to Immobilon-P (Millipore) and probed with the antibodies by the methods of Baek et al.(6) . Antibody cross-reactivity of proteins was visualized with chemiluminescence reagent (DuPont NEN) using Kodak XAR-5 film. GTP binding ability of the partially purified expressed proteins (50 ng/tube) was measured by GTP-mediated inhibition of the TGase activity in the presence and absence of GTP. Partial purification of the expressed proteins was achieved by Q-Sepharose chromatography. The lysates (5 mg of protein) of transfected COS-1 cells were solubilized with 0.4% sucrose monolaurate (SM) at 4 °C for 1 h. The extracts were applied to a Q-Sepharose column (0.4 ml) equilibrated with a buffer containing 20 mM Hepes, pH 7.4, 1 mM EGTA, 0.5 mM dithiothreitol, 70 mM NaCl, and 0.05% SM. The columns were washed with 3-5 ml of 170 mM NaCl in the same buffer. The hhGalpha(h) and its mutant proteins were eluted with 500 mM NaCl in the same buffer. The yields were analyzed by immunoblotting using G antibody and Coomassie Blue staining following SDS-polyacrylamide gel electrophoresis. The TGase activity of the purified proteins was determined in the presence of 0.5 mM CaCl(2) and 1 mM dithiothreitol by evaluating [^3H]putrescine incorporation into N,N`-dimethyl casein(1, 2) .

Coimmunoprecipitation

For coimmunoadsorption of PLC or the alpha(1)-adrenoreceptor with hhGalpha(h) and its mutant proteins, G antibody-protein A-agarose was prepared according to the method of Schneider et al.(15) . The membranes (150 µg/sample) in HSD buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.5 mM dithiothreitol) were solubilized with 0.4% SM in the presence of 50 µM GTPS and 2 mM MgCl(2) at 4 °C for 1 h. The extracts were incubated with the antibody-agarose or preimmune-agarose (30 µl suspension/assay) at 4 °C for 1 h. The resins were collected by centrifugation at 2000 rpm for 15 min and washed three times with HSD buffer containing 5 µM GTPS, 1 mM MgCl(2), and 0.05% SM. The antibody resin-bound PLC activity was measured using 50 µM CaCl(2) for 20 min at 30 °C in a 100-µl final volume. The PLC activity absorbed to preimmune-protein A-agarose was negligible and taken as a nonspecific binding. Inhibition of the coimmunoprecipitation of PLC by peptides was determined using the membrane extracts, which were preincubated with peptides. The samples were incubated with the antibody-agarose (30 µl/tube) in the presence of 50 µM GTPS and 2 mM MgCl(2) at 4 °C for 2 h. The resin-bound PLC was determined under the same conditions as described above. The phosphatidylinositol 4,5-bisphospate was 100 µM (1200 cpm/nmol) in the assay.

For the coimmunoprecipitation of the alpha(1)-adrenoreceptor with hhGalpha(h) and its mutants, the membranes that coexpressed hhGalpha(h) or its mutants with alpha-adrenoreceptor or alpha-receptor alone were incubated with 5 times 10M(-)-epinephrine at 4 °C for 3 h. The membranes were then solubilized with 0.2% SM in HSD buffer in the presence of 5 times 10M(-)-epinephrine at 4 °C for 1 h. The recovery of proteins in the extracts usually reached 40% for the receptor and 40-50% for hhGalpha(h) and its mutants (see also (5) ). Since the hhGalpha(h) and its mutants were overexpressed 3-5-fold as compared to the alpha(1)-adrenoreceptor, the membrane extracts (100 fmol of the alpha(1)-receptor) were incubated with 100 µl of G antibody-agarose or nonimmune sera-agarose in 300 µl (final volume) with gentle rotation at 4 °C for 2 h. After centrifugation at 2000 rpm, the alpha-receptor density (50 µl of supernatant/tube) was measured using 3 nM [^3H]prazosin (final) in the presence or absence of 10M phentolamine in 200 µl (final volume) after removing excess(-)-epinephrine through a dried 3-ml Sephadex G-25 column. The preimmune sera-agarose-treated samples were used as controls to calculate the amounts of the receptor immunoprecipitated for each sample.

Other Assays

PLC activity was evaluated by the method of Im et al.(8) . The alpha(1)-adrenoreceptor density was determined using the radiolabeled alpha(1)-specific antagonist [^3H]prazosin(5) . The alpha-adrenoreceptor-activated PLC stimulation was determined after normalizing receptor number. The protein concentration was determined by the method of Bradford(16) .


RESULTS AND DISCUSSION

The isolated full-length hhGalpha(h) cDNA was an exact match in the nucleotide and deduced amino acid sequences with the human endothelial TGase II(14) . To identify interaction sites of hhGalpha(h) with the alpha(1)-adrenoreceptor and PLC, systematic 10-amino acid-deleted mutants of hhGalpha(h) cDNA(s) were generated from the C-terminal end (Fig. 1). The full-length hhGalpha(h) cDNA and truncated hhGalpha(h) cDNA(s) were cotransfected into COS-1 cells with alpha-adrenoreceptor cDNA. The expressed proteins were recognized by the G antibody as well as guinea pig TGase II antibody and were of the expected sizes with 80 kDa for full-length hhGalpha(h) and a decrease in size as the length of nucleotide deletion increased (Fig. 2A). The alpha-receptor was also expressed, resulting in 2-3 pmol/mg protein of [^3H]prazosin binding. The expressed hhGalpha(h) proteins exhibited both GTP binding and TGase activities (Fig. 2B and inset). The Ca-stimulated TGase activities of expressed hhGalpha(h) and its truncated mutants were completely inhibited with >100 µM GTP, and the inhibitory potency (IC) of GTP was in the range of 20-50 µM for all hhGalpha(h) proteins, also confirming that GTP is a negative regulator for the TGase of Galpha(h)(1, 3, 4) .


Figure 1: Map of the 687-amino acid hhGalpha(h) chain and designation of truncation mutants and hhGalpha(h)/human FXIIIa chimera in hhGalpha(h) polypeptide.




Figure 2: A, an autoradiogram of an immunoblot of the expressed membrane proteins from COS-1 cells transfected with hhGalpha(h) or mutant hhGalpha(h) polypeptides using polyclonal G antibody. Molecular sizes (in kilodaltons) are shown on the left. Endogenous Galpha(h) (TGase II) is seen in the membranes from expressed alpha-receptor (alpha-AR) alone and mutants. The protein expression level was 4-10-fold higher than the endogenous Galpha(h). B, the GTP-mediated inhibition of TGase activities of the expressed hhGalpha(h) and mutants. The inhibition of TGase activity (50 ng/tube) was determined with GTP in the presence of 0.1 mM [^3H]putrescine, 1% N,N`-dimethyl casein, 0.5 mM CaCl(2), and 2 mM MgCl(2) in HSD buffer at 30 °C for 30 min. The inset shows the stimulation of TGase (50 ng/tube) in the presence of 0.5 mM CaCl(2) without GTP under the same conditions. The results are the mean of three independent experiments performed in duplicate.



All mutants, as well as hhGalpha(h), elevated the basal PLC activity as compared to that of the alpha-receptor alone (Fig. 3A), indicating induction of precoupled protein complexes resulting from overexpression of the proteins(1, 18) . The(-)-epinephrine-mediated activation of PLC was increased approximately 2-fold with hhGalpha(h) and the DeltaK676 mutant, whereas the DeltaL656 and DeltaE646 mutants lost the agonist-mediated PLC stimulation, exhibiting a level similar to that of the alpha-receptor alone. The DeltaN666 mutant stimulated PLC upon activation of the alpha(1)-receptor, but to a lesser extent than wild type. These data suggested that a region comprising 20 amino acids between His and Lys is critical for coupling to the alpha-receptor or PLC.


Figure 3: Coupling ability of expressed hhGalpha(h) and its mutants. A, epinephrine-stimulated inositol 1,4,5-triphosphate (IP) accumulation in membranes from COS-1 cells coexpressed with alpha-receptor and hhGalpha(h) or its mutants. The alpha(1)-receptor-mediated PLC stimulation was determined after normalizing receptor number (100 fmol/tube) in a 100-µl final volume. Receptor number was normalized, since the receptor number is the determinant in signal manipulation, not G-protein number(17) , and the expression level of hhGalpha(h) and its mutants was also 3-5-fold higher than the receptor level. The results are the mean ± S.E. of three independent experiments performed in duplicate. alpha-AR, alpha-adrenoreceptor; Ep, (-)-epinephrine; Ph, phentolamine. B, remaining alpha-adrenoreceptor in the supernatants after coimmunoprecipitation with hhGalpha(h) and its mutants using G antibody-protein A-agarose. The alpha(1)-adrenoreceptor absorbed to preimmune protein A-agarose was less than 5% as compared to the resin-untreated samples. The preimmune-resin-treated samples were taken as 100% for each sample. C, immunoadsorption of a complex of PLC with hhGalpha(h) and its mutants by G antibody-protein A-agarose. The PLC activity absorbed to preimmune protein A-agarose was negligible and taken as nonspecific binding. The data shown are the mean ± S.E. of three independent experiments in triplicate.



The possibility that the receptor and/or PLC recognition sites were deleted was examined by coimmunoprecipitation. The mutants, DeltaK676, DeltaN666, and DeltaL656, coexpressed with the alpha-adrenoreceptor, coimmunoprecipitated >90% alpha(1)-receptor as wild type did, indicating that the receptor interaction site on these mutants was intact (Fig. 3B). However, the mutant, DeltaE646, consistently coimmunoprecipitated less receptor (80%) than other mutants but more than the receptor alone (35%). Although less coimmunoprecipitation of the receptor with this mutant suggested that this region on hhGalpha(h) might contain the receptor interaction site, this point should be further investigated. Coimmunoprecipitation of the receptor with membrane extract from the expressed alpha-receptor alone was probably due to complex formation between the internal Galpha(h) and the receptor.

The loss of PLC interaction site was then assessed by coimmunoprecipitation (Fig. 3C). The results revealed that the DeltaK676 mutant coimmunoprecipitated PLC as effectively as the wild type, whereas the DeltaL656 and DeltaE646 mutants failed to coimmunoprecipitate PLC, showing a similar level to that of the alpha-receptor alone. The DeltaL666 mutant again showed lower coimmunoprecipitation of PLC than hhGalpha(h) but higher than theDeltaL656 and DeltaE646 mutants. The loss of the PLC interaction site was further confirmed by determining PLC stimulation in response to GTP (Fig. 4, A and B). As expected, the basal levels of PLC in membranes expressing hhGalpha(h) and mutants were increased 36-fold compared to the alpha-receptor alone. Within these increases, the PLC basal activity gradually decreased as the deletion size increased (Fig. 4A). In the presence of GTP, the expressed hhGalpha(h) and the mutant DeltaK676 increased PLC stimulation 2-fold (Fig. 4B). Increases in deletion size also resulted in a gradual decrease of GTP-mediated PLC stimulation. Mutants DeltaL656 and DeltaE646 lost ability to stimulate PLC in response to GTP. These results were consistent with the finding from the coimmunoprecipitation studies and strongly suggested that a region between His and Lys on hhGalpha(h) contained a PLC interaction site.


Figure 4: The GTP-mediated PLC activation. The membranes (30 µg/tube) were preincubated with and without 0.1 mM GTP in the presence of 2 mM MgCl(2) at 30 °C for 30 min. Production of IP(3) was measured with various concentrations of CaCl(2) at 30 °C for 10 min. Panels A and B are shown in the absence and presence of GTP, respectively. The data shown are the mean ± S.E. of three independent experiments in duplicate.



To further define this putative PLC interaction site, four overlapping peptides corresponding to the deleted regions of hhGalpha(h) were synthesized and tested for their ability to inhibit coimmunoprecipitation of PLC (Fig. 5A). Peptide 4 (Leu-Lys), among the four peptides, was able to inhibit coimmunoprecipitation of PLC (Fig. 5B). Coimmunoprecipitation of PLC was inhibited in a concentration-dependent manner, and at 100-200 µM of the peptide, the inhibition reached 80%, suggesting that other interaction site(s) probably exist (Fig. 5C). The competition potency (IC) of peptide 4 for the interaction between hhGalpha(h) and PLC was 20 µM.


Figure 5: A, map of the synthesized peptides of hhGalpha(h). Overlapping amino acids in peptides 3 and 4 are indicated with asterisks. B, effect of peptides of hhGalpha(h) on coimmunoprecipitation of PLC by G antibody-protein A-agarose. The membrane extracts (100 µg/tube) were preincubated with 100 µM peptide and subjected to immunoprecipitation. PLC activity was determined as detailed under ``Experimental Procedures.'' The results are a mean ± S.E. of three independent experiments performed in duplicate. C, competition of peptide 4 with hhGalpha(h) to coimmunoprecipitate PLC by G antibody-protein A-agarose. The experiments were performed with various concentrations of peptide 4 under the same conditions as detailed under ``Experimental Procedures.'' The data presented are a mean of the duplicated experiments using three independently expressed proteins.



The findings that a region of 12 amino acids between Leu and Lys in hhGalpha(h) contains a PLC interaction site were refined by a chimera, hhGalpha(h)/FXIIIa, in which eight amino acid residues Val-Lys of hhGalpha(h) were substituted with the corresponding region (Ile-Ser) of human factor XIIIa (see Fig. 1)(13) . This region of FXIIIa was chosen because FXIIIa does not interact with or stimulate PLC or bind GTP(4) , and this region of FXIIIa is distinct among TGases(13) . The chimera was expressed 7-fold higher than endogenous Galpha(h) and to the similar level of hhGalpha(h) (Fig. 6A). The chimera protein exhibited GTP binding and TGase activity at the same levels as the wild type (Fig. 6B). In addition, using the partially purified chimera when the GTP-mediated inhibition of TGase activity was titrated, the inhibition was similar to DeltaK676 and wild type, indicating that substitution of this region did not change GTP binding affinity (data not shown). The chimera also failed to stimulate PLC in response to GTP (Fig. 6C) and upon activation of the alpha-receptor (data not shown). The G antibody did not coimmunoprecipitate PLC but effectively coimmunoprecipitated the receptor (data not shown). These findings clearly demonstrate that the C-terminal region of hhGalpha(h) from Val to Lys is a critical site for interaction and stimulation of PLC.


Figure 6: Coupling ability of the expressed hhGalpha(h)/FXIIIa chimera. A, an autoradiogram of an immunoblot of the expressed membrane proteins from COS-1 cells transfected with alpha(1)-adrenoreceptor alone or with wild-type or hhGalpha(h)/FXIIIa chimera. alpha-AR, alpha-adrenoreceptor; WT, hhGalpha(h); Chi, hhGalpha(h)/FXIIIa chimera. B, TGase and GTP binding activity of the expressed wild-type or hhGalpha(h)/FXIIIa chimera in the membranes. Both enzyme activities were determined in the presence or absence of 0.1 mM GTP as detailed in Fig. 2B. C, GTP-mediated PLC stimulation. The PLC activity was measured using the membranes (30 µg/tube) in the presence of 0.1 mM GTP, 2 mM MgCl(2), and 10 µM CaCl(2) under the conditions detailed under ``Experimental Procedures.'' The results are the mean ± S.E. of three independent experiments performed in duplicate. IP, inositol 1,4,5-triphosphate.



The substituted region of the chimera has a significant change in properties of the amino acids (Fig. 1). Thus, four charged amino acids (Asn, Glu, Asp, and Lys) were substituted for serine, except Asp. Hydrophobic amino acids (Val, Val, and Phe) were also changed to smaller (V666A and F668M) or larger (V665I) amino acids. Although it has been suggested that replacement of a bulky side chain of hydrophobic amino acids can result in loss of activity due to unfavorable van der Waals interactions(19) , overall hydrophobicity of the substituted amino acids, however, remains similar. Therefore, it is unlikely that a hydrophobic interaction is responsible for coupling of hhGalpha(h) to PLC. Three charged amino acids in this region, i.e. a hydrophilic interaction, probably play a critical role in the contact of hhGalpha(h) with PLC.

In heterotrimeric G-protein-mediated signaling systems, the near C-terminal domain of the alpha-subunit appears to contain an effector contact region (adenylyl cyclase with Galpha(s)(20, 21, 22) and cGMP-phosphodiesterase with Galpha(t)(23) ). Despite extensive primary structural differences between Galpha(h) and alpha-subunits of the heterotrimeric G-proteins, our data indicate that Galpha(h) seems to share this common structural feature in signaling. Our data also suggest that the carboxyl domain of Galpha(h) with its primary structure distinct from other transglutaminases is likely to be involved in signaling functions, including receptor and GTP binding sites.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1GM45985. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Cardiology (FFB-37), Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-8860; Fax: 216-444-8372.

(^1)
The abbreviations used are: TGase II, transglutaminase II; FXIIIa, coagulation factor XIIIa; GTPS, guanosine 5`-O-(3-thiotriphosphate); PCR, polymerase chain reaction; PLC, phospholipase C; SM, sucrose monolaurate; hhGalpha(h), human heart Galpha(h).


ACKNOWLEDGEMENTS

We thank Dr. D. Perez for alpha-adrenoreceptor cDNA clone and Dr. P. J. Birckbichler for the monoclonal antibody of guinea pig transglutaminase II. We also thank Drs. E. Plow and S. Karnik for valuable discussions.


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