N-terminal Short Sequences of alpha  Subunits of the G12 Family Determine Selective Coupling to Receptors*

Yoshiaki YamaguchiDagger, Hironori Katoh, and Manabu Negishi§

From the Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received for publication, February 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The Galpha subunits of the G12 family of heterotrimeric G proteins, defined by Galpha 12 and Galpha 13, have many cellular functions in common, such as stress fiber formation and neurite retraction. However, a variety of G protein-coupled receptors appear to couple selectively to Galpha 12 and Galpha 13. For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via Galpha 12 and Galpha 13, respectively. We recently showed that active forms of Galpha 12 and Galpha 13 interact with Ser/Thr phosphatase type 5 through its tetratricopeptide repeat domain. Here we developed a novel assay to measure the activities of Galpha 12 and Galpha 13 by using glutathione S-transferase-fused tetratricopeptide repeat domain of Ser/Thr phosphatase type 5, taking advantage of the property that tetratricopeptide repeat domain strongly interacts with active forms of Galpha 12 and Galpha 13. By using this assay, we identified that thrombin and LPA selectively activate Galpha 12 and Galpha 13, respectively. Galpha 12 and Galpha 13 show a high amino acid sequence homology except for their N-terminal short sequences. Then we generated chimeric G proteins Galpha 12N/13C and Galpha 13N/12C, in which the N-terminal short sequences are replaced by each other, and showed that thrombin and LPA selectively activate Galpha 12N/13C and Galpha 13N/12C, respectively. Moreover, thrombin and LPA stimulate RhoA activity through Galpha 12 and Galpha 13, respectively, in a Galpha 12 family N-terminal sequence-dependent manner. Thus, N-terminal short sequences of the G12 family determine the selective couplings of thrombin and LPA receptors to the Galpha 12 family.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Heterotrimeric G proteins act as a molecular switch that conveys signals from G protein-coupled receptors (GPCRs)1 in the cell membrane to intracellular downstream effectors, and they are divided into four major families, Gs, Gi, Gq, and G12, based on sequence homology of their alpha  subunits (1). The G12 family is defined by Galpha 12 and Galpha 13, and they share 67% amino acid sequence identity (2). Active forms of Galpha 12 and Galpha 13 induce several biological responses via activation of Rho, a major downstream common target for Galpha 12 and Galpha 13, including stimulation of stress fiber formation and focal adhesion assembly (3), induction of neurite retraction (4), induction of apoptosis (5), transformation of fibroblasts (6), activation of phospholipase D (7), inhibition of Ca2+-dependent exocytosis (8), and activation of c-Jun N-terminal kinase (9). Recently, several proteins that interact with both Galpha 12 and Galpha 13 have been identified, including p115, a Rho guanine nucleotide exchange factor (10), cadherin (11), and radixin (12). Therefore, Galpha 12 and Galpha 13 are thought to transduce mutual downstream signaling through these effectors. In contrast, a variety of GPCRs appear to selectively couple to Galpha 12 or Galpha 13 (13). For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via Galpha 12 and Galpha 13, respectively, by using dominant negative mutants of Galpha 12 and Galpha 13 (13). However, this receptor-G protein coupling is not directly evaluated.

Recently, we demonstrated that Galpha 12 and Galpha 13 interact with Ser/Thr phosphatase type 5 (PP5) through its tetratricopeptide repeat (TPR) domain and stimulate its phosphatase activity (14). In this study, we developed a novel assay to evaluate the activities of Galpha 12 and Galpha 13 by using glutathione S-transferase (GST)-fused TPR domain of PP5 (GST-TPR), taking advantage of the property that GST-TPR strongly interacts with active forms of Galpha 12 and Galpha 13. By using this assay, we showed selective activations of Galpha 12 by thrombin and Galpha 13 by LPA, and found that N-terminal short sequences of Galpha 12 and Galpha 13 determine these selective activations.

    EXPERIMENTAL PROCEDURES
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Materials-- Agents obtained and commercial sources were as follows: thrombin and LPA, Sigma; rabbit polyclonal anti-Galpha 12 and anti-Galpha 13 antibodies and mouse monoclonal anti-RhoA antibody, Santa Cruz Biotechnology, Inc.; horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin G antibody, DAKO; and Chemiluminescence ECL Western blotting system, Amersham Biosciences. GST-fused proteins were purified from Escherichia coli as described previously (8, 14). The TPR domain used for GST-TPR is a splicing variant of rat PP5 that contains residues 9-122 following a new short coding region (12 amino acids; SRALGMGQLPAP) (14). The nucleotide sequence of this TPR variant is available from GenBankTM/EMBL/DDBJ under accession number AB101661.

Plasmid Constructions-- cDNAs of wild-type Galpha 12 and Galpha 13 (Galpha 12WT and Galpha 13WT), and their constitutively active mutants of Galpha 12 (Galpha 12Q229L, Galpha 12QL) and Galpha 13 (Galpha 13Q226L, Galpha 13QL) were obtained as described previously (4, 14). To generate chimeric cDNA of Galpha 13 substituted with the N-terminal short sequence of Galpha 12 (Galpha 12N/13C), cDNAs encoding the N-terminal part of Galpha 12 (amino acids 1-37) and the C-terminal part of Galpha 13 (amino acids 31-377) were combined by using a BglII site generated by PCR-mediated mutagenesis, and then it was subcloned into BamHI/NotI sites of pcDNA3 vector. Chimeric cDNA of Galpha 12 substituted with the N-terminal short sequence of Galpha 13 (Galpha 13N/12C) was generated by combining cDNAs encoding the N-terminal part of Galpha 13 (amino acids 1-30) and the C-terminal part of Galpha 12 (amino acids 38-379), using a XbaI site generated by PCR-mediated mutagenesis, and it was subcloned into HindIII/NotI sites of pcDNA3 vector.

Cell Culture, Transfection, and Measurement of Activities of Galpha 12, Galpha 13, and RhoA-- 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 4 mM glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin under humidified air containing 5% CO2 at 37 °C. 1 × 106 cells were seeded in a 60-mm dish and transfected with 2 µg of cDNA using LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. After transfection, medium was replaced to Dulbecco's modified Eagle's medium containing 1% fetal bovine serum and incubated for 12-15 h. To examine the activities of alpha  subunits, cells transfected with Galpha 12WT, Galpha 12QL, Galpha 13WT, Galpha 13QL, Galpha 12N/13C, or Galpha 13N/12C were stimulated with either thrombin (1.5 units/ml) or LPA (1 µM) for the indicated times, rinsed once with phosphate-buffered saline, and lysed with 500 µl of the ice-cold cell lysis buffer (20 mM Hepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) containing 3 µg of GST-TPR. In the case of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> treatment, cells treated with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (50 µM AlCl3, 10 mM MgCl2, and 5 mM NaF) for 3 h were lysed with cell lysis buffer containing AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. Cell lysates were then centrifuged for 5 min at 12,000 × g at 4 °C, and the supernatants were incubated with glutathione-Sepharose beads for 30 min at 4 °C. After the beads were washed with the ice-cold cell lysis buffer, the bound proteins were eluted in Laemmli sample buffer and analyzed by 10% SDS-PAGE and immunoblotting with anti-Galpha 12 and anti-Galpha 13 antibodies (1:200 dilution, both). To examine the endogenous RhoA activity, cells transfected with Galpha 12WT, Galpha 13WT, Galpha 12N/13C, or Galpha 13N/12C were stimulated with either thrombin (1.5 units/ml) or LPA (1 µM) for 1 min, and cell lysates were incubated with GST-fused Rho-binding domain of Rhotekin as described previously (8), and bound proteins were immunoblotted with anti-RhoA antibody (1:100 dilution). The primary antibodies were detected by using horseradish peroxidase-conjugated secondary antibodies and the ECL detection kit. Densitometric analyses were performed by using NIH Image software, and the amounts of active forms of alpha  subunits and RhoA were normalized to the total amounts of alpha  subunits and RhoA in cell lysates, respectively.

    RESULTS AND DISCUSSION
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We reported previously that active forms of Galpha 12 and Galpha 13, but not Galpha q and Galpha i2, interact with PP5 through its TPR domain (14). To develop a method to measure the activities of Galpha 12 and Galpha 13, we examined the ability of GST-TPR to pull-down active forms of Galpha 12 and Galpha 13. As shown in Fig. 1, GST-TPR strongly precipitated Galpha 12WT and Galpha 13WT in the presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, an activator of the Galpha subunits (15), and their constitutively active forms, Galpha 12QL and Galpha 13QL, but precipitation of Galpha 12WT and Galpha 13WT in the absence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> was almost negligible. These results demonstrate that active forms of Galpha 12 and Galpha 13 can be detected by the GST-TPR pull-down assay.


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Fig. 1.   Pull-down assay for active forms of Galpha 12 and Galpha 13 by using GST-TPR. 293T cells were transfected with an empty vector (lanes 1 and 5) or with a vector for Galpha 12WT (lanes 2 and 3), Galpha 12QL (lane 4), Galpha 13WT (lanes 6 and 7), or Galpha 13QL (lane 8). Cells were treated (lanes 3 and 7) or not treated (lanes 1, 2, 4, 5, 6, and 8) with AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> for 3 h before lysis. Cell lysates were incubated with GST-TPR, and the bound alpha  subunits and total amounts of alpha  subunits in cell lysates were determined by immunoblotting with polyclonal antibodies against Galpha 12 (lanes 1-4) and Galpha 13 (lanes 5-8). The results shown are representative of three independent experiments that yielded similar results.

Thrombin and LPA have been shown to induce stress fiber formation via Galpha 12 and Galpha 13, respectively, by using dominant negative mutants of Galpha 12 and Galpha 13 (13). Therefore, we measured the thrombin and LPA-induced Galpha 12 and Galpha 13 activations by using this GST-TPR pull-down assay. Because the expression levels of endogenous Galpha 12 and Galpha 13 were extremely low in a variety of cell lines including 293T, COS-7, and Swiss 3T3 cells (Fig. 1 and data not shown), it was unable to detect the endogenous activities of Galpha 12 and Galpha 13 by using GST-TPR pull-down assay. Then we transfected 293T cells with Galpha 12WT or Galpha 13WT and treated the cells with either thrombin or LPA. Thrombin rapidly activated Galpha 12WT, the activation level reaching a maximum at 1 min and gradually decreasing to the basal level within 10 min. However, LPA had no effect on Galpha 12WT activity (Fig. 2A). On the other hand, LPA rapidly activated Galpha 13WT, whereas thrombin had no effect on Galpha 13WT activity (Fig. 2B). Thus, thrombin and LPA receptors selectively couple to and activate Galpha 12 and Galpha 13, respectively.


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Fig. 2.   Selective activations of Galpha 12 by thrombin and Galpha 13 by LPA. 293T cells transfected with an expression vector for Galpha 12WT (A) or Galpha 13WT (B) were treated with thrombin (1.5 units/ml) or LPA (1 µM) for the indicated times. The cell lysates were incubated with GST-TPR, and the amounts of active forms of Galpha 12 and Galpha 13 and total amounts of Galpha 12 and Galpha 13 in cell lysates were determined by immunoblotting with polyclonal antibodies against Galpha 12 and Galpha 13 (insets in A and B), respectively. Galpha 12 (A) and Galpha 13 (B) activities are indicated by the amounts of active forms of Galpha 12 and Galpha 13 normalized to the amounts of Galpha 12 and Galpha 13 in whole cell lysates, respectively, and values are expressed as fold of the value of ligand-untreated cells (at time 0 min). The results shown are the means ± S.E. of triplicate experiments.

Galpha 12 and Galpha 13 show a high amino acid sequence homology (67%) except for their N-terminal short sequences in which the amino acid identity is only 16% (2). This limited homology prompted us to speculate that the N-terminal short sequences determine the specificities of Galpha 12 and Galpha 13 for thrombin and LPA, respectively, and then we generated chimeric alpha  subunits Galpha 12N/13C and Galpha 13N/12C, in which the N-terminal sequence is replaced each other (Fig. 3A). Because anti-Galpha 12 and anti-Galpha 13 antibodies used here are raised against their N-terminal amino acids, Galpha 12N/13C and Galpha 13N/12C can be recognized with respective antibodies. 293T cells expressing Galpha 12N/13C or Galpha 13N/12C were treated with either thrombin or LPA, and activities of chimeric proteins were measured by GST-TPR pull-down assay. Similar to Galpha 12, Galpha 12N/13C was strongly activated by thrombin but not by LPA (Fig. 3, B and D), though it is mostly composed of Galpha 13. On the other hand, Galpha 13N/12C, which is mostly composed of Galpha 12, was strongly activated by LPA but not by thrombin (Fig. 3, C and E). These results indicate that N-terminal short sequences of Galpha 12 and Galpha 13 determine their specificities for thrombin and LPA, respectively.


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Fig. 3.   N-terminal domains of Galpha 12 and Galpha 13 determine the selective activations by thrombin and LPA. A, diagrams of Galpha 12N/13C and Galpha 13N/12C. The numbers of corresponding amino acids are also shown. B and C, 293T cells transfected with an expression vector for Galpha 12N/13C (B) or Galpha 13N/12C (C) were treated with thrombin (1.5 units/ml) or LPA (1 µM) for 1 min. The cell lysates were incubated with GST-TPR, and the amounts of active forms of Galpha 12N/13C and Galpha 13N/12C and total amounts of Galpha 12N/13C and Galpha 13N/12C in cell lysates were determined by immunoblotting with polyclonal antibodies against Galpha 12 (B) and Galpha 13 (C), respectively. D and E, quantification of the effects of thrombin and LPA on Galpha 12N/13C (D) and Galpha 13N/12C (E) activities. The Galpha 12N/13C and Galpha 13N/12C activities are indicated by the amounts of active forms of Galpha 12N/13C and Galpha 13N/12C normalized to total amounts of Galpha 12N/13C and Galpha 13N/12C in whole cell lysates, and values are expressed as fold increase over the value of cells that were not treated with either thrombin or LPA. Results shown are the means ± S.E. of triplicate experiments.

Activation of Galpha 12 or Galpha 13 increases RhoA activity (8, 16). To next evaluate whether selective couplings of thrombin and LPA receptors to Galpha 12 and Galpha 13 are functional, we examined the effects of thrombin and LPA on RhoA activity in the cells expressing Galpha 12, Galpha 13, or chimeric alpha  subunits. Thrombin and LPA activated RhoA strongly via Galpha 12WT and Galpha 13WT, respectively (Fig. 4A), the specificities corresponding with the results shown in Fig. 2. With respect to chimeric alpha  subunits, thrombin and LPA activated RhoA strongly via Galpha 12N/13C and Galpha 13N/12C, respectively (Fig. 4, B and C). Thus, thrombin and LPA receptors functionally couple to Galpha 12 and Galpha 13, respectively, and these functional couplings are determined by the N-terminal short sequences of Galpha 12 and Galpha 13.


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Fig. 4.   Stimulation of RhoA activity by thrombin and LPA through N-terminal-dependent couplings to Galpha 12 and Galpha 13, respectively. A and B, 293T cells transfected with an empty vector, or a vector for Galpha 12, Galpha 13, Galpha 12N/13C, or Galpha 13N/12C were treated with thrombin (1.5 units/ml) or LPA (1 µM) for 1 min. The cell lysates were incubated with GST-fused Rho-binding domain of Rhotekin, and the amounts of active form of RhoA and total amounts of RhoA in cell lysates were determined by immunoblotting with a monoclonal antibody against RhoA. Galpha 12, Galpha 13, Galpha 12N/13C, and Galpha 13N/12C in cell lysates are also shown. C, quantification of the effects of thrombin and LPA on RhoA activities via Galpha 12N/13C and Galpha 13N/12C. Lanes 1-9 correspond with those in panel B. The RhoA activity is indicated by the amounts of active form of RhoA normalized to total amounts of RhoA in whole cell lysates, and values of RhoA activity are expressed as fold increase over the value of empty vector-transfected cells that were not treated with either thrombin or LPA. Results shown are the means ± S.E. of triplicate experiments.

Gohla et al. (3) showed that LPA stimulated [alpha -32P]GTP azidoanilide-labeling of both Galpha 12 and Galpha 13 in the isolated membranes, and Ponimaskin et al. (17, 18) showed that thrombin also stimulated binding of [35S]GTPgamma S to both Galpha 12 and Galpha 13 in the membranes, indicating that thrombin and LPA receptors have the ability to stimulate both Galpha 12 and Galpha 13 in vitro. However, thrombin- and LPA-induced stress fiber formations via RhoA are mediated specifically by Galpha 12 and Galpha 13, respectively (3, 13), suggesting that both receptors selectively use G proteins in intact cells. In this study, we developed a novel assay to evaluate the activities of Galpha 12 and Galpha 13. By using this assay, we directly demonstrated that thrombin and LPA selectively activate Galpha 12 and Galpha 13, respectively, in intact cells. These results taken together indicate that thrombin and LPA receptors can potently couple to both Galpha 12 and Galpha 13 in vitro, but they show selective usage of G proteins in vivo. We further demonstrated that N-terminal short sequences of Galpha 12 and Galpha 13 determine the selective couplings of thrombin and LPA receptors to Galpha 12 and Galpha 13, respectively. C-terminal domains of alpha  subunits are well known to play an important part in specifying receptor interactions of G proteins (1). However, Galpha 12 and Galpha 13 share very high amino acid sequence identity in the C-terminal region (2), suggesting that C-terminal domains of Galpha 12 and Galpha 13 do not contribute to the coupling selectivity of thrombin and LPA receptors.

Recently, Waheed et al. (19) showed that Galpha 12 resides in lipid raft fraction, whereas Galpha 13 is not associated with lipid rafts in COS-7 cells and NIH 3T3 cells, and that the N-terminal sequence of Galpha 12 including a palmitoylation site is important for the lipid raft localization of Galpha 12. Moreover, they showed that the targeting of Galpha 12 to lipid raft is mediated by Hsp90, a chaperone that specifically associates with Galpha 12 but not Galpha 13 (20). Although intracellular localizations of thrombin and LPA receptors are not yet known, selective targeting of G proteins and receptors to either lipid raft fraction or other fraction may determine the selective coupling. Galpha 12 and Galpha 13 mostly display mutual downstream signal transductions, such as RhoA activation and serum response factor activation (2, 16). Recently, a variety of downstream effectors or interacting proteins of the Galpha 12 family have been identified, including Rho guanine nucleotide exchange factors, cadherin, and PP5, and they interact with both Galpha 12 and Galpha 13 (10, 11, 14), suggesting that downstream signal transduction pathways of Galpha 12 and Galpha 13 are highly overlapped. On the other hand, stress fiber formations induced by a variety of GPCRs are selectively mediated by either Galpha 12 or Galpha 13 (13), suggesting that receptor-G protein coupling is selective. Galpha 12 and Galpha 13 are highly expressed in brain, and it has been shown that Galpha 12 is mainly expressed in somata of the neurons, but Galpha 13 is predominantly localized in the neuropil of central neurons, indicating that Galpha 12 and Galpha 13 exert their effects at the different area in the cell (21). In the light of this knowledge, different localizations of receptors and G proteins in the cells may be important for their selective coupling.

In conclusion, we have developed a novel assay to evaluate the activities of Galpha 12 and Galpha 13, and we directly demonstrated selective couplings of thrombin and LPA receptors to Galpha 12 and Galpha 13, respectively, dependent on the N-terminal short sequences of G proteins. This study will contribute not only to the understanding of the activation mechanism of the Galpha 12 family, but will also help to elucidate the signaling mechanism of heterotrimeric G proteins.

    FOOTNOTES

* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Takeda Science Foundation.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.

Dagger Recipient of Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists.

§ To whom correspondence should be addressed: Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4547; Fax: 81-75-753-7688; E-mail: mnegishi@pharm.kyoto-u.ac.jp.

Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M301409200

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; PP5, Ser/Thr phosphatase type 5; TPR, tetratricopeptide repeat; GST, glutathione S-transferase; GST-TPR, GST-fused TPR domain of PP5; WT, wild-type.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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