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,4
* Department of Pharmacology and Toxicology, and
UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294; and
** Department of Mathematics and Statistics, Auburn University, Auburn, AL 36849
4To whom correspondence should be addressed. E-mail: Coral{at}uab.edu.
ABSTRACT
Genistein, the primary isoflavone component of soy, consumed in the diet during the prepubertal period only, and the combined prepubertal and adult periods, suppresses chemically induced mammary cancer in rats. Gestational or adult-only exposures do not provide protection. An inverse relation exists between cancer susceptibility and mammary gland differentiation. The current study used proteomic technology to investigate genistein mechanisms of action as related to programming against chemically induced mammary cancer. Rats were injected subcutaneously with 500 µg genistein/g body weight on d 16, 18, and 20 postpartum. At d 21, mammary glands were subjected to 2-dimensional polyacrylamide gel electrophoresis. After gel scanning, image analysis, and MS, 6 proteins were determined to be differentially regulated and identified. One protein, GTP-cyclohydrolase 1 (GTP-CH1), was confirmed as being significantly upregulated at d 21 by immunoblot analysis. Investigation of downstream signaling from GTP-CH1 showed that tyrosine hydroxylase was upregulated and vascular endothelial growth factor receptor 2 (VEGFR2) was downregulated in the mammary glands of 50-d-old rats treated with genistein in the prepubertal period. This and previous work suggest that early prepubertal exposure to genistein enhances cell proliferation by upregulating GTP-CH1 and the epidermal growth factor (EGF)-signaling pathway, and hence cell differentiation and gland maturation. This unique developmental maturation leads to a new biochemical blueprint, whereby the cells have reduced EGF signaling and VEGFR2, which renders the mature mammary gland less proliferative and less susceptible to cancer. This study demonstrated the usefulness of proteomics for the discovery of novel pathways that may be involved in cancer prevention.
KEY WORDS: • 2-D gel • genistein • GTP-CH1 • VEGFR2 • proteomics
Inherited genetic risk for breast cancer accounts for <10% of all breast cancer cases (1). Epidemiological reports and data from laboratory studies suggest that approximately three-fourths of all cancer deaths are attributable to lifestyle factors, indicating that environment and diet must play a role in the cause and prevention of cancer. Many cancer causation and prevention studies involving environmental chemicals have investigated direct effects in adult animals, because cancer is considered a disease associated with aging. However, exposure to environmental chemicals during critical periods of early development plays an important role in breast cancer susceptibility in adulthood (2).
Our laboratory has focused research on the chemopreventative nature of the soy isoflavone genistein. We demonstrated that prepubertal exposure to genistein decreases tumor multiplicity and diminishes incidence of adenocarcinomas in the dimethylbenz[a]anthracene (DMBA)5 model of mammary cancer. Whole-mount analysis of mammary glands showed that prepubertal exposure to genistein enhances mammary gland differentiation, that is, results in fewer terminal end buds and more lobules (3,4). Terminal end buds are the least mature terminal ductal structures and are the most susceptible to chemical carcinogens (5). The resulting morphological action of genistein can be characterized as enhancing gland maturation. This maturation yields a gland that is ultimately less proliferative and less susceptible to chemical carcinogenesis (3,4,6,7). In general, the morphological development of the mammary gland extends beyond the embryonic stage and largely occurs during postnatal development. The postnatal development of the rat mammary gland closely resembles that of the human and provides a meaningful model for studying cancer chemoprevention (8).
To enhance our ability to discover biochemical pathways that genistein may affect in vivo, we adopted the technologies of 2-dimensional (2-D) gel electrophoresis and MS to find new biomarkers of genistein action in the rat mammary gland. In the present study, we discovered, via 2-D gel analysis, differential protein expression of GTP-cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), a necessary cofactor for catecholamine synthesis and nitric oxide production (9). We also constructed a possible mechanism of chemoprevention by changes in the downstream enzyme tyrosine hydroxylase (responsible for dopamine production) and a possible target of its action, vascular endothelial growth factor receptor 2 (VEGFR2), and showed how these changes in protein expression can be involved in the chemopreventative action of genistein.
Methods and materials
Animals. Animal care and treatments were performed according to established guidelines, and protocols were approved by the UAB Animal Care Committee. Animals were housed in a temperature-controlled facility with a 12-h light:dark cycle. Female Sprague-Dawley rats (Charles River) were fed phytoestrogen-free AIN-76A pellets (Harlan Teklad) (10). Breeding was conducted in our facility, and animals were separated as soon as sperm or plugs were detected. Pregnant animals were caged separately. Offspring were collected and sexed on d 1 postpartum, and all litters were adjusted to 10 offspring (5 males and 5 females). Offspring were weaned at d 21 postpartum. Female offspring were assigned to the following groups: vehicle (dimethylsulfoxide; Sigma), genistein (98.5% purity; Hoffmann-LaRoche), estradiol benzoate (EB; Sigma), or daidzein (Hoffmann-LaRoche). On postnatal d 16, 18, and 20, female offspring were injected subcutaneously with either 500 µg genistein/g body weight (BW), 500 µg daidzein/g BW, 500 ng EB/g BW, or an equivalent volume of the vehicle. The genistein dose was previously shown to enhance mammary gland maturation and suppress chemically induced mammary cancer development (3). The daidzein dose was the same as the genistein dose, and the EB dose results in a similar uterotrophic effect as the dose of genistein (2,4,11). Animals were killed on d 21 or 50 postpartum (prepubertal and young adult rats, respectively). At the time of death, the 2 abdominal mammary glands were excised and snap-frozen at –80°C for later analysis. Three unique sets of genistein- and control-treated animals were generated for these experiments. Set 1 was used for the production of 2-D gels (5 rats/group); set 2 was used for validation studies via immunoblot analysis (8–10 rats/group). Set 3 included the daidzein and EB treatment groups (8 rats/group) as the weakly estrogenic isoflavone and estrogen-positive controls, respectively.
2-D gels. Frozen mammary tissues (n = 5, genistein; n = 5, control) were pulverized and homogenized in lysis buffer as described by Fountaoulakis et al. (12). Protein concentrations of the individual samples were determined using the BioRad protein assay reagent (BioRad) in 96-well microtiter plates. From each sample, 150 µg total protein was loaded onto 11-cm immobilized pH gradient strips, pH 3 to 10 (GE Healthcare). The samples were allowed to rehydrate into the gels overnight at room temperature. The next day, strips were placed onto a flatbed electrophoresis unit (Multiphor II; GE Healthcare) for 1st-dimension focusing. The voltage gradient was 500 V for 1 h, followed by a ramp to 3500 V for 1.5 h, and sustained at 3500 V for 22 h. After the 1st dimension was completed, the strips were equilibrated using 100 mmol/L dithiothreitol for 30 min followed by 30 min equilibration with 120 mmol/L iodacetimide. After equilibration, isoelectric focusing gels were placed on a 1.5-mm, 12.5% SDS vertical gel (Criterion; BioRad), and the gels were assayed on a vertical electrophoresis unit (Dodecacell, BioRad) at a constant 200 V. When the assays were completed, the gels were fixed for 1 h in 40% methanol and 10% acetic acid (v:v), and stained overnight with Sypro Ruby gel stain (Molecular Probes). Gels were destained (10% methanol, 7.5% acetic acid; v:v) for a minimum of 4 h. Stained gels were scanned via a Perkin Elmer ProExpress densitometer and initially analyzed with the Progenesis 2-D gel software system (Nonlinear).
MS.
For identification, selected protein spots were manually excised from the gels. Gel plugs were destained, dried in a SpeedVac (Savant), rehydrated, and digested with trypsin (Roche). Samples were mixed 1:10 (v:v) with a saturated solution of sinapinic acid in 50% aqueous acetonitrile and 0.1% aqueous trifluoroacetic acid (1:1, v:v), and 1 µL was spotted onto the stainless steel matrix-assisted laser desorption ionization (MALDI) target plate and allowed to dry before analysis by MALDI–time-of-flight (TOF) MS. Peptide molecular ions were analyzed in linear positive-ion mode using a Voyager Elite MS (Applied Biosystems). Using an acceleration voltage of 25 kV and a laser intensity of 2500 V, each spot was analyzed a minimum of 3 times, accumulating spectra composed of 
200 laser shots in total. The resulting spectra were analyzed by DataExplorer (Applied Biosystems). The instrument was calibrated using an external apomyoglobin standard and internally using a trypsin autolysis peak. Resulting spectra were baseline corrected and filtered for noise. MALDI-TOF MS–produced spectra were analyzed using the MASCOT program (13).
Immunoblot analysis. Mammary gland samples (n = 8/treatment) were homogenized in radioimmunoprecipitation lysis buffer (Upstate Biotech) with protease inhibitors (2 mmol/L Na vanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, 2 µg leupeptin/mL, 2 µg aprotinin/mL) while rotating at 4°C. Proteins from each sample (20 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (BioRad). Membranes were blocked and incubated with the appropriate antibody, including polyclonal rabbit antirat GTP-CH1 (Dr. Gregory Kapotos, Wayne State University), and the commercially available tryosine hydroxylase, tryptophan hydroxylase, phenylalanine hydroxylase, inducible nitric oxide synthase, and VEGFR2 (Santa Cruz). Membranes were subsequently incubated with appropriate secondary antibody conjugated to horseradish peroxidase for detection after reaction with a chemiluminescent substrate (Pierce). Membranes were either exposed to autoradiography film for band quantitation by densitometric analysis or directly analyzed using a VersaDoc 4000 (BioRad). Densitometric analysis was performed using QuantityOne software (BioRad).
ELISA. Dopamine levels were evaluated using an ELISA kit (Rocky Mountain Diagnostics) according to the manufacturer’s protocol. Briefly, whole mammary glands were homogenized in RIPA buffer with protease inhibitors; 10 µL from each sample was used per evaluation, and all samples were analyzed in duplicate in 96-well microtiter plates. The range of standards was from 0 to 2560 µg/L, with a total of 6 separate concentrations. Samples were evaluated using an Ultrospec 2000 UV/Visible spectrophotometer (GE Healthcare, formerly Pharmacia Biotech).
Immunohistochemisty (IHC). Immunolocalization of tyrosine hydroxylase, GTP-CH1, and VEGFR-2 was performed as described in Brown et al. (14) on trypsinized paraffin sections of mammary glands fixed in 4% paraformaldehyde by a modified avidin-biotin complex technique. Primary antibodies were the same as described for the immunoblot methods plus mouse monoclonal dopamine (Abcam). All primary antibodies were incubated overnight at 4°C in a humidity chamber with rocking. The secondary antibodies were biotinylated goat antirabbit IgG (Vector Laboratories). Negative control slides were treated with preimmune serum and omission of primary antibody with each batch of slides. Slides were evaluated on a Nikon Labphot-2 (Nikon) at 200x magnification (10x ocular and 20x objective).
HPLC. BH4 levels were assayed using HPLC methods described by Fukushima and Nixon (15). Briefly, whole mammary glands (n = 10/treatment) were lysed in 0.15 mol/L HCLO4 and 0.1 mol/L H3PO4. An internal standard of D-erythro-neopterin (gift of Dr. Wayne Kapatos, Wayne State University) was added to each sample. To each sample 300 µL of a 1% iodine and 2% potassium iodide solution was added, and the samples were incubated in the dark for 1 h. A solution of 1% ascorbic acid was then added to stop the reaction. Proteins were precipitated, and the supernatant was passed through a Dowex 50W-X4 column (Bio-Rad). Pteridines were eluted from the column using 1 mol/L NH4OH directly onto a Dowex AG1-X8 column (Bio-Rad). Finally, pteridines were eluted using 1 mol/L acetic acid, then dried by evaporation. The samples were analyzed on a Perkin Elmer Series 200 autosampler and pump with a Shimadzu RF-551 spectrofluorometric detector using an Alltech Associates Spherisorb ODS-1 4.6-mm-i.d. x 250-mm column. The mobile phase was 5% methanol in water at 1 mL/min.
Statistics. For 2-D gels, the average values for normalized spot volume were compared using the Student’s t test. Average values of immunoblots for each treatment group were compared using ANOVA combined with Tukey’s test (SAS) and reported as percentage of the control.
Results
Proteins from the mammary glands of 21-d-old rats with prepubertal exposure to genistein or control treatments were evaluated using 2-D gel electrophoresis. After image analysis, 88 proteins common to all gels were selected for further statistical evaluation. Based on normalized spot volume, the expression of 6 protein spots was found to differ significantly between the genistein-treated and the control animals (P < 0.05) (Fig. 1). Table 1 lists the protein names and their descriptive roles(16–20). For 5 of these proteins (B–F), we were able to obtain antibodies and pursue protein confirmation. Immunoblot analysis of the individual proteins confirmed only the change observed for GTP-CH1 (Fig. 2).
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BH4 levels were measured by HPLC (Fig. 3). Although mean values for BH4 from the mammary glands of treated and control animals did not differ significantly, the overall variance in expression as evaluated using the Weibel model was significantly different (P < 0.05).
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Discussion
We previously reported that short-term treatment of rats during the prepubertal period with genistein can confer a long-term protection against chemically induced mammary cancer (2–4,6). The cellular mechanism of action was enhanced mammary gland maturation. Until recently, few biochemical mechanisms have been associated with this mammary cancer chemoprevention model (14). Hence, we embarked on the use of 2-D gels for finding additional differentially expressed proteins and MALDI-TOF MS for identifying these proteins.
Our first endeavor into discovery proteomics revealed 6 protein spots from 2-D gels that were differentially expressed in the mammary glands of 21-d-old rats treated with genistein in the prepubertal period. However, only protein expression change for GTP-CH1 was confirmed by immunoblot analysis. GTP-CH1 expression was upregulated in the mammary glands of 21-d-old but not in 50-d-old rats with only prepubertal exposure to genistein. This demonstrates a direct and reversible action, because GTP-CH1 levels were unchanged in the mammary glands of the resulting adult rats.
To determine the biochemical significance of GTP-CH1 upregulation in the immature mammary gland, we investigated downstream metabolic pathways (Fig. 8). GTP-CH1 is the rate-limiting enzyme in the production of BH4. The latter is an essential cofactor for the enzymes of catecholamine and nitric oxide syntheses. Catecholamines, in particular, can signal through membrane-bound adrenergic receptors to affect differentiation and development of cells (23). In PC12 cells, stimulation by epidermal growth factor or nerve growth factor increases cell proliferation through elevation of intracellular BH4 (24). BH4 is also implicated in cell proliferation on the basis of studies using murine erythroleukemia cells as a model for erythrogenesis (25). Although we did not find a direct correlation between GTP-CH1 levels and BH4 levels, we did notice a difference in the overall regulation of BH4 between the 2 groups, based on variance. This is not conclusive evidence of the actions of GTP-CH1, but others, such as Vann et al. (26), have reported a divergence in the regulation of BH4 and enzymes that require it (primarily nitric oxide synthase in their experiment).
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Given the significant increase in GTP-CH1 expression and the potential involvement of its downstream targets, we investigated whether the direct effects of genistein altered the expression of the catecholamine synthesis enzymes. Immunoblot analysis showed that only the level of tyrosine hydroxylase was significantly altered. Specifically, we found that it was upregulated in mammary glands of 50- but not 21-d-old rats. Notably, this change in protein level at 50 d occurred in the absence of the original effector, genistein, and was present even though the significant difference in the level of GTP-CH1 no longer existed. This is suggestive of a programming effect on protein expression, that is, a developmental modification with no observable effect on the protein immediately after treatment or exposure but a delayed manifestation in adult life (2). In light of this change, we turned our attention to how downstream metabolic pathways and signaling of tyrosine hydroxylase in the mature gland could be involved in suppressing cancer in rats with only prepubertal exposure to genistein.
Teunis et al. (29) examined the role of dopamine sensitivity in tumor growth. They found that rats with high dopaminergic activity had a reduction in tumor size compared with rats with low dopaminergic reactivity. Associating elevated dopamine levels with suppressed mammary tumorigenesis, they noted that the angiogenic response to VEGF could be inhibited by administration of dopaminergic agonists. Basu et al. (30) reported that dopamine acts through the dopamine 2 receptor to induce the endocytosis of VEGFR2 and thereby inhibit or prevent VEGF binding, receptor phosphorylation, and subsequent signaling steps. They reported that immunohistochemical studies did not find tyrosine hydroxylase–positive nerves in tumors, and the dopamine concentration in malignant tumors was significantly reduced compared with concentrations in controls. Ferguson et al. (31) reported that lifetime exposure to genistein (500 µg/g) can potentiate dopamine levels in striata of amphetamine-exposed animals. This demonstrates a lasting effect of genistein that does not affect the baseline levels but allows for adjustments in the face of certain perturbations. In addition, researchers found that genistein decreased both transcription and protein levels of VEGF and that this decrease is involved in the loss of angiogenesis (32,33). Hefelfinger et al. (34) demonstrated that inhibition of VEGFR2 will prevent DMBA-induced mammary tumors in rats.
Given a potential link among tyrosine hydroxylase, VEGFR2, and angiogenesis, we conducted analysis for the VEGFR2 protein. The results showed a significant decrease in VEGFR2 expression in the mammary glands of 50-d-old rats with only prepubertal genistein treatment. These results complemented the finding that tyrosine hydroxylase levels were elevated in the mammary glands of 50-d-old rats with prepubertal genistein treatment. We speculated that upregulation of tyrosine hydroxylase results in dynamic upregulation of catecholamines, which, in turn, decrease the VEGFR2 levels, resulting in decreased ability to promote angiogenesis (Fig. 8). Ortega et al. (35) implicated VEGFR2 in mediating cell proliferation; therefore, a decrease in VEGFR2 may decrease the overall proliferative potential of the mammary gland. The absence of a demonstrable change in dopamine concentrations may mean that the concentration is dynamic or that changes in concentration within the microenvironment may not manifest in detectible or significant change in the larger sample (whole mammary gland).
Having established possible mechanisms by which the direct effects of genistein on GTP-CH1 and the programming effects of genistein on tyrosine hydroxylase could lead to developmental changes in the mammary glands and alter susceptibility to breast cancer, we deemed it important to determine whether the observed effects resulted from actions unique to genistein or were part of a broader estrogenic response. To answer this question, we investigated how selected proteins respond in animals exposed to estrogen or the other major soy isoflavone, daidzein. Results from the present study showed that prepubertal exposure to EB and daidzein did not produce any significant changes for GTP-CH1, tyrosine hydroxylase, VEGFR2, or any of the other evaluated proteins in the mammary glands of 21- and 50-d-old rats. This suggested that genistein exhibits some selective effect independent of estrogen action. The lack of significant changes brought about by daidzein as compared with genistein supports the role of these changes as unique potential mechanisms for genistein chemoprevention.
Localization of the proteins of interest to the same cell type may provide greater insight into the role of these proteins within this model. Our IHC data indicate that the proteins of interest (GTP-CH1, tyrosine hydroxylase, and VEGFR2) appear to be modulated in their expression only in the lining of epithelial cells. This is important because it is hypothesized that the epithelial cell type may be the origination of tumor cells. Because all 3 proteins are expressed in the same cell type and IHC confirmed that these changes were similar to those observed for immunoblot analysis, this may indicate a paracrine or an autocrine regulatory action. This colocalization will deserve further examination as we explore the temporal expression of these proteins in this model.
Although we were able to find several proteins determined to be differentially regulated using 2-D gel analysis, our confirmation attempts using immunoblots found that only one of these proteins was significantly changed by genistein treatment. This lack of cohesion between our 2-D gels and immunoblotting analysis may be because of stringent specificity of site-directed antibodies and/or due to the low number of samples used for the generation of the 2-D gel data set. We used n = 5 for the 2-D gels, but our confirmation was with n = 8/group. Given that the sensitivity of the immunotechniques should be much greater than that of the staining techniques, we recognize that we must increase the number of samples in subsequent 2-D gel studies to decrease this false-positive rate. However, we recognize that a finite number of samples can be reasonably assayed to produce quality data without exhausting resources. Also, the use of t-tests and other parametric procedures may not be appropriate mechanisms for evaluating the 2-D gel data set; therefore, alternative testing strategies may be needed for evaluating potential subtle changes in expression. These evaluations may focus on general changes of protein expression rather than specific changes related to mean expression. For these reasons, we are still interested in pursuing the other identified proteins.
In summary, data from this and previous work suggest that early postnatal (prepubertal) exposure to genistein enhances cell proliferation by upregulating GTP-CH1 and the EGF-signaling pathway and hence cell differentiation and gland maturation. This unique developmental maturation leads to a new biochemical blueprint, whereby the cells have reduced EGF-signaling and VEGFR2, which renders the mature mammary gland less proliferative and less susceptible to chemically induced mammary cancer initiation, angiogenesis, and cancer progression. Therefore, genistein acts through a diverse and coordinated effect of signaling mechanisms and pathways that likely account for the cellular changes responsible for its chemopreventive action.
Because cancer is often thought of as a disease of aging, we have to understand and appreciate that the mammary gland undergoes multiple stages of development. The use of animal models allows us to follow the changes within the gland to determine how the actions of genistein may alter the morphological characteristics as well as to examine how these morphological changes may work at the biochemical level to yield sustained changes that affect susceptibility. Two-dimensional gel exploration looks at the most common and persistent changes within the cellular populations. Knowledge gained from these experiments provides more information on the possible mechanisms of action of genistein that result in chemoprevention. Specifically, this report is the first to identify GTP-CH1 protein alterations in the mammary gland as well as their potential regulation by genistein. Future work will examine other members of these signaling pathways more closely and address the potential mechanisms involved.
ACKNOWLEDGMENTS
We thank Gregory Kapatos at Wayne State University, Detroit, MI, for providing the GTP-CH1 antibody, and Stephen Barnes and Landon Wilson at the UAB Comprehensive Cancer Center Mass Spectrometry Core Facility for their assistance with MS protein identification. We thank Isam-Eldin Eltoum for his assistance with the histomorphology and Jun Wang for general technical assistance. As well, we thank Lori Coward at Southern Research, Birmingham, AL, for measurement of BH4 levels.
FOOTNOTES
1 Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 14–15, 2005. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by (in alphabetical order) California Avocado Commission; California Walnut Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.; The Hormel Institute; National Fisheries Institute; The Solae Company; and United Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R. Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman are employed by conference sponsor American Institute for Cancer Research; V.L.W. Go, no relationships to disclose. 
2 Author disclosure: No relationships to disclose.
3 The research was supported by grants from the U.S. Department of Defense (DOD BC 17-03-1-0433) and the National Institutes of Health (NIH U01 ES 012771-02). CR was supported by stipends from the U.S. Department of Defense (DOD BC 17-00-1-0119) and the Susan G. Komen Foundation (DISS0201242). The mass spectrometers used to generate the proteomic data were purchased using funds provided by Shared Instrumentation Grants (S10 RR11329 and S10 RR13795) from the National Center for Research Resources and an award from the UAB Health Services Foundation General Endowment Fund. The UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility is supported by a core grant (P30 CA-13148) from the National Cancer Institute.
5 Abbreviations: 2-D, 2-dimensional; BH4, tetrahydrobiopterin; BW, body weight; DMBA, dimethylbenz[a]anthracene; EB, estradiol benzoate; EGF, epidermal growth factor; GTP-CH1, GTP-cyclohydrolase 1; IHC, immunohistochemisty; MALDI-TOF, matrix-assisted laser desorption ionization–time of flight; MAPK, mitogen-activated protein kinase; VEGFR2, vascular endothelial growth factor receptor 2.
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-synuclein; C, ABC transporter; D, peroxiredoxin 1; E, 14–3-3 
; F, GTP-CH1.
