F11 neuroblastoma cell - Interaction of Heterotrimeric Go Protein with Protein Kinase A

F11 cell shows the characteristics of rat dorsal root ganglion (DRG) and mouse neuroblastoma N18TG-2. F11 cell expresses δ-opioid, prostaglandin and bradkinin receptor which involved in immune system and dihydropyridine-sensitive calcium ion channels. In differentiation process, F11 cell also synthesized and released a substance P-like compound.

Generally, F11 cell could be induced to differentiate into neuron in the presence of cAMP and prostaglandin.

- 9 - 5. Purpose

This study was performed to find the binding site of Goα in interaction with PKA-Cα and Goα, and to identify the importance of interaction through the GTPase domain of Goα in differentiation of F11 cell. Co-immunoprecipitation and GST pull down assay results demonstrate that the Goα protein interacts with PKA-Cα through the GTPase domain of Goα.

In the differentiation of F11 cell, the neurite number of F11 cell was increased in the presense of interaction with Goα and PKA-Cα through the GTPase domain of Goα.

Consequently, the function of Goα as Scaffold protein of PKA-Cα in subcellular is key mechanism for differentiation of F11 cell.

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II. MATERIALS AND METHODS

1. Cells

F11 cells are hybrid cell line from rat dorsal root ganglion (DRG) and mouse neuroblastoma cell line of N18TG-2 offered from Dr. M. Fishman (Harvard University, Cambridge, MA, U.S.A.). The 293T and F11 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin.

2. Plasmids

The Goα, Giα and chimeric proteins (Goα/iα, Giα/oα) with a FLAG-tag at the N terminus were previously described (Ghil et al., 2006). The pcFLAG-Goα plasmid was created by digesting with EcoRI and XbaI from pRC/CMV-Goα. The pcFLAG-Giα plasmid was also created by enzyme digestion with XbaІ and ApaІ from original pBluescript SK (+)-Giα. In case of pcFLAG-Goiα and Gioα, digested GTPase domains by BamHI enzyme were switched each other. All FLAG and GST-Goα deletion constructs except for GST-GoαΔG2, GST-GoαΔNHLG1 were offered from Dr. Ghil (Kyong-gi University, Suwon, South Korea).

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Fig. 3. Amino acid sequence anaylsis of Goα and Giα. Goα shares 71% amino acid with Giα. Arrow indicates starting point of GTPase domain.

3. Manufacture of GST-fusion protein

To make the GST-GoΔG2, PCR products of the rest domain except for G2 domain was obtained from the backbone of GST-Goα (full length) using forward primer and reverse primer. Subsequently, T-vector (Promega, Madison, WI, USA) was ligated with PCR products. Final digested fragment (N, H, L and G1 domain, 645bp) by EcoRI inserted into pGEX-2T expression vector.

Templete: GST-Goα

Expression vector: PGEX-2T

Forward primer: 5’ GGA ATT CGA TTC ATG GGA 3’

Reverse primer: 5’ TCA GTG GAT CCA CTT CTT AC 3’

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Fig. 4. GST-GoΔG2. GST-GoΔG2 has all domain of Goα except for G2 part. The fragment (645bp) by EcoRI was inserted into PGEX-2T expression vector. The MCS (multiple cloning sites) of GST-GoΔG2 is located in below the construct map.

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To make the GST-GoΔNHLG1, the only G2 domain was obtained from the backbone of GST-Goα using BamHI and EcoRI enzyme. Digested fragment (G2 domain, 428bp) inserted into pGEX- 5X-3 expression vector.

Templete: GST-Goα

Expression vector: PGEX-5X-3 Enzyme digestion: BamHI, EcoRI

Fig. 5. GST-GoΔNHLG1. The digested fragment (428bp) from GST-Gαo with BamHI and EcoRI was inserted into PGEX-5X-3 vector. The MCS (multiple cloning sites) of GST-GoΔNHLG1 is located in below the construct map.

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4. Establishment of stable F11-Goα, Giα, Goiα, Gioα cell line

To make 4 types of F11 stable cell line, 5×10⁴ F11 cells were plated in 24well. After 1 day, each of the FLAG-tagged DNA (Goα, Giα, Goiα, Gioα) was transfected into F11 cell using CaPO₄ transfection method following the manufacturer’s instructions. Subsequently, G418 600 μg/ml was added in trasfected 4 types of F11 cell to select the positive cell for over 7 days. Because the pcDNA3.1 expression vector of FLAG-tagged DNA expresses the neomycin resistant gene.

5. Cell culture

The neuroblastoma F11 cell is hybrid cell that has characteristics of rat dorsal root ganglion (DRG) and mouse neuroblastoma NT18TG-2 (M. Fishman, Harvard University, U.S.A). The F11 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) that contains 10% FBS (fetal bovine serum, Gibco) and mixture of 100 U/ml penicillin and 100 μg/ml streptomycin in condition of 5% CO₂ in air at 37 °C.

6. Differentiation

After removing the culture media, the F11 cells were washed with HBSS one time.

Finally, the differentiation media that includes 0.5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 30 μM forskolin (Sigma, St. Louis, MO)was treated in F11 cells which were attached on coated cover glass using poly-D-lysine (0.1 mg/ml, Sigma). F11 cells were maintained in differentiation media for 2 days.

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7. Quantification of the number of neurite and protrusion

Neurite means the process that has the longer length than cell body diameter. Each neurite was individually extended from the cell body was used to count in differentiated F11 cell through eyes, directly. Protrusion means all extended process from cell body regardless of cell body diameter length. Protrusion was also counted through eyes.

8. Co-immunoprecipitation

293T cells were plated in growth media of 100 mm dish with a density of 2×10⁶cell. Next day, 293T cells were transiently transfected with FLAG-tagged Goα, Giα, Goiα, Gioα and PKA-Cα (pcDNA3-Cα)by CaPO₄method. When the cells are almost filled with 100 mm dish about 80%, the dish was gently washed 2 times with ice cold-PBS. And harvested cell was lysed with PBTX Buffer. For pre-clearance, protein A Sepharose CL-4B beads (50%

slurry, GE Healthcare) added to the soluble 293T cell extracts. After washing, Five hundred micrograms of protein dissolved in 500 μl of PBTX was incubated with 1 μg of antibody against Goα or Cα (Santa Cruz) with gentle rotation for 4 hr at 37 °C (or O/N at 4 °C). After 2 hr incubation with 50 μl of beads, beads were repeatly washed with PBXT buffer five times. Finally, the remaining PBXT buffer was removed, perfectly. The bound proteins were eluted with SDS sample buffer and subjected to immunoblot analysis by using indicated antibodies (anti-Cα, anti- FLAG).

- 17 - 9. GST pull down assay

The deletion constructs of Goα were inserted to pGEX-2T (vector of GST-GoΔG2, GE Healthcare, Wisconsin, USA) & pGEX-5X-3(vector of GST-GoΔNHLG1, GE Healthcare) expression vector. From transformed Escherichia coli BL21 cell population, a peaked colony inoculated in 3 ml TB (Terrific Broth) growth medium for one day. Subsequently, 10~200 μl of suspension which contains BL21 cells was inoculated 10~200 ml new TB growth medium again at 30 °C for about 12 hr until the ratio of OD is reached 0.6~1 at OD600. Finally, IPTG (final 0.1 mM concentration, Isopropyl-β-D-thio-galactoside, USB) was addedto growth media and incubated at 30 °C for 20 hr. Bacterial cell lysates which were extracted from harvested E. coli BL21 cells were incubated with Glutathione Sepharose 4B beads (Pharmacia. Biotech, USA) for 1 hr at 4 °C in PBTX buffer (PBS containing 1% TritonX-100, 5 mM MgCl₂, 1 mM EDTA, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, and 2 mM Phenylmethylsulfonyl fluoride). After washing extensively 5 times with PBTX buffer. Purified PKA-Cα (Promega) was added to precipitated GST fusion protein that contains beads and incubated for 1 hr at 4 °C. After washing with PBTX buffer, the bound proteins were eluted with SDS sample buffer and subjected to immunoblot analysis by using antibodies against against PKA-Cα (Santa Cruz, CA, U.S.A).

10. Immunocytochemistry

The 5×10³ F11 cells were placed on cover slip that was coated with Poly D-lycine (0.1 mg/ml). The cover slips were fixed in cold methanol or 4% paraformaldehyde (BBC Biochemical) for 10 min at room temperature. Subsequently, to permeabilize, cells were

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treated with the 0.1% TPBS (in PBS containing 0.1% Triton X-100) for 10 min. After blocking with 10% BSA and 1% normal goat serum (NGS) for 1 hr at room temperature, The cells were incubated with both polyclonal rabbit anti-FLAG (Sigma) and monoclonal mouse anti-Tuj-1 (Covance, Richmond, CA) primary antibody to labels overexpressed Goα and neurites at 4 °C for Over Night. Subsequently, Cells were washed with 0.1%TPBS three times and treated with second antibody Alexa 568 goat rabbit IgG, Alexa 488 goat anti-mouse IgG (Molecular Probes, Willow, OR, USA) and Hoechest (1: 5000~10000, Sigma) Images were captured using fluorescence microscope.

11. Immunoblotting

293T cells (F11 cell), in 100 mm dish of 80~90% (50~60%) confluency were washed with a ice cold-PBS (phosphate buffered saline) two times. The harvested cells in ice PBXT buffer 1 ml (0.5 ml) were incubated in rotation rocker, gently at 4 °C for 1 hr. The isolated protein sample by centrifuge (12000 rpm, 10 min, 4 °C) was quantified through the Bradford assay. Subsequently, the equal amounts of protein were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were incubated in 5% skim milk blocking solution in TTBS (20 mM Tris-HCl (PH7.5), 150 mM NaCl, and 0.1% Tween 20) for 1 hr at room temperature. And the membranes were incubated with the primary monoclonal anti-FLAG (Sigma, 1:500), polyclonal anti-Goα (Santa Cruz, 1:500) or polyclonal PKA-Cα (Santa Cruz, 1:500) in 1X PBS for 2 hr at room temperature or overnight at 4 °C. After washing with TTBS buffer three times, the membranes were treated with the second

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antibody of horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (Vector, 1:500~1:10000), rabbit IgG (Zemed, 1:10000~1:20000). After washing with TTBS buffer three times, the proteins were visualized by enhanced chemiluminescence (ECL) method.

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III. RESULTS

1. Goα protein directly interacts with PKA-Cα through the GTPase domain of Goα Generally, unlike Giα, Goα does not inhibit adenylyl cyclase. Therefore, Goα protein is likely to have no influence in adenylyl cyclase-related signaling pathway. But, the previous study has shown that α subunit of Go protein is involved in cAMP triggered signaling pathway (Ghil et al., 2000). Presumably, Goα protein may regulate PKA signaling pathway, directly. Subsequently, the recent study demonstrates that Goα directly binds to free catalytic subunit of PKA that was dissociated from holoenzyme but not PKA-RПβ (Ghil et al., 2006).

Subsequently, in our study, the interaction of PKA-Cα and Goα was also confirmed through co-immunoprecipitation experiment using Goα, Giα, the two chimeric protein of Go/iα containing Goα (1-213) and Giα (213-354), Gi/oα containing Giα (1-212) and Goα (214-354) (Fig. 1A). The result reveals that Goα directly binds to PKA-Cα but not Giα (Fig. 1B).

Additionaly, to investigate subdomain of Goα protein that interacts with PKA-Cα, we performed GST pull-down assay with many different types of GST fusion protein that contain a partial deletion form of Goα domain (Fig. 2A). GST-Goαconsists of five domains:

N: n-terminal domain, H: heliex domain, L: linker I, II domain and G: GTPase domain as G1, G2. In case of GST-GoαΔG2, it has all subdomain except for G2 domain that is a part of the GTPase domain at direction of C-terminal. On the contrary, GST-GoαΔNHLG1 has only G2 domain. GST-GoαΔG as control of GoαΔG2 does not have the whole GTPase domain of Goα. And GST-GoαΔNH as control of GoαΔNHLG1 has linker and GTPase domain. As a

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result, all GST fusion proteins which contain GTPase domain of Goα bound to PKA-Cα not GST-GoαΔG. But, the binding affinity of GoαΔG2 is lower than any other proteins, relatively.

Consequently, the result reveals that GTPase domain of Goα is important binding subdomain in interaction with PKA-Cα (Fig. 2B).

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Fig. 6. Goα protein directly binds to PKA-Cα, not Giα. (A) Schematic presentation of Goα (black square), Giα (white square), two chimeric Goα/iα (complex of black/white) and Giα/oα (complex of white / black). In case of chimeric proteins, among five domains, a partial domain of GTPase domain of Goα and Giα was shifted each other. (B) Co-immunoprecipitation result. 293T cell extracts overexpressing four proteins and PKA-Cα were immunoprecipitated with polyclonal anti-Cα antibody. The precipitated proteins were immunoblotted with anti-FLAG antibody to identify which proteins can be binded to PKA-Cα. Note that Goα, 2 chimeric protein bound to PKA-Cα except for Giα.

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GST pull down

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Fig. 7. Goα protein directly interacts with PKA-Cα through the GTPase domain of Goα.

(A) Schematic presentation of Goα and deletion constructs which have GST-tag at N-terminus. GST-GoαΔG, ΔNH is the control of GoαΔG2, ΔNHLG1. (B) GST pull down assay result. Only GST-GoαΔGmutant does not bind to purified PKA-Cα. There is no GTPase domain of Goα in GST-GoαΔG mutant. Binding affinity of GST-GoαΔG2 is lower than GST-GoαΔNHLG1, relatively. (C) Coomassie blue staining. There is a difference among the five constructs in protein expression level. Although protein expression level of GST-GoαΔG is relatively more elevated than any other constructs, it does not bind to PKA-Cα. GTPase domain of Goα is important part in combination with PKA-Cα.

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2. The interaction of Goα and PKA-Cα has an influence on neurite formation of F11 cell

According to previous studies, Go protein is highly expressed in brain and enriched at neuronal growth cone of neurites. A report demonstrates that Go protein is the most abundantly expressed in brain of central nervous system (CNS) plays crucial role in neuronal development (Dr Ghil et al., 2008). Moreover, in field of differentiation and neuritogenesis, a study reveals that Go protein was produced at the tip of the growing neurite during neuronal differentiation and the expression level of Go protein was elevated during neuronal differentiation in fusion cells of neuroblastoma and glioma (Mullaney and Milligan et al., 1989). Another study discusses about overexpression of Go can modulate neuritogenesis in PC12 (Strittmatter et al., 1994) and Neuro2A (Jordan et al., 2005) cells.

To define a specific role of Goα protein in cell level, we examined influence of Goα and Go mutant types in F11 cell. It was known that the number of neurite in F11 cell overexpressing Goα^WT and Goα^Q205L was increased, whereas the average length of neurite was decreased (Ghil et al., 2000). On the evidence of previous studies, we performed the differentiation experiment of F11 cell overexpressing another Goα deletion constructs (GoΔLG, GoΔNHL) to prove the fact that GTPase domain of Goα is essential of an increase in neurite number of F11 cell. We transiently transfected into F11 cell using Goαand deletion constructs of Goα; GoΔLG which was deleted the GTPase domain of Goα, GoΔNHL which has only GTPase domain of Goα (Fig. 3A). As a result, two neurites which extending from cell body were formed as bipolar formation in normal F11 cell. But overexpressing Goα- F11 cell has many neurites which were extended from cell body at 2 days. The number of neurite

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per cell was determined as extended thing from the cell body. In case of the GoΔLG, the extending neurites are the same as normal F11 cell. On the other hand, the neurites of F11 cell overexpressing GoΔNHL seems to be different as compared with F11 cell (Fig. 2B).

Therefore, we directly counted total neurite number through the eyes to more precise analysis. Quantitative analysis result indicates that the neurite number per F11 cell overexpressing Goα protein which has GTPase domain was increased than normal F11 cell.

But, overexpressing GoΔLG-F11 cell was nearly similar to normal F11 cell. Because PKA-Cα can’t bind to GoΔLG which was deleted the GTPase domain of Goα. The neurite number of GoΔNHL overexpressing F11 cell was a little bit decreased rather than the control (F11 cell) although it has the GTPase domain of Goα (Fig. 2C). Presumably, GoΔNHL has extremely only GTPase domain without the other subdomain as compared with GoΔLG, the action of GoΔNHL may be functionally restricted. Thus, binding ability and activity can be decreased.

Consequently, the interaction of Goα and PKA-Cα through the GTPase domain of Goα clearly affect to neurite formation of F11 cell that was differentiated by cAMP. Therefore, the increased neurite number of F11 cell was caused by the interaction of overexpressed Goα and PKA-Cα. Overexpression of Goα can functionally affects to differentiation system of F11 cell by PKA signaling pathway.

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Fig. 8. GTPase domain of Goα is essential for an increase in neurite number. (A)The Go deletion constructs; GoΔLG which was deleted the GTPase domain of Goα, GoΔNHL which has only GTPase domain of Goα. (B) Immunocytochemistry of F11 cell. The neurite number of F11 cells which were transiently transfected with several FLAG-tagged constructs is different from each other (normal F11, Go, GoΔLG, GoΔNHL). All of neurites were stained with anti-tuj-1 (2^nd Alexa 488 green). The anti-FLAG (2^nd Alexa 568 red) labeled overexpressed FLAG-tagged Go, GoΔLG, GoΔNHL. Hoechst (blue) dye stained nucleus.

Merged cells were used to count the neurite number of FLAG positive cell (white arrow head). Scale bar 50 μM. (C) Quantitative analysis of neurite number per cell. Individually, normal F11 cell (N=148) and all selected FLAG positive cells of two group were counted.

(Go-N=46, Go∆LG-N=17, Go∆NHL-N=10). Especially, FLAG-Gopositive F11 cell has many neurites than normal F11 and Go∆LG-F11 cell. FLAG-Go∆LG and F11 cell are equal in number. In case of FLAG-Go∆NHL positive cell, the neurite number was decreased rather than normal F11 cell slightly.

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3. The neurite formation was changed depending on differentiation time

The one of the major features of F11 neuroblastoma cell is that forskolin or db-cAMP promotes neurite outgrowth during differentiation. And the average neurite length was grown in a concentration-depend manner (Ghil et al., 2000). In here, to observe the effect of Goα, Giα, Goiα and Gioα on morphological change of F11 cells during differentiation in real time, 30 μM forskolin which stimulates adenylyl cyclase was treated for 2 days. Total 48 hr (2 days) were divided into 4 groups of 12 hr, 24 hr, 36 hr and 48 hr. For this experiment, each different types of stable F11 cell (Goα, Giα, Goiα, Gioα) line were newly produced using G418 selection system. The result indicates that the cell morphology of all stable F11cell is similar in initial state of differentiation up to 24 hr. But at 36 hr, the cell morphology was distinctly different from each other. In here, we observed many protrusions which extending from cell body. The concept of protrusion has a short or equal length than cell body diameter is different from neurite standard. In later, protrusion will be generated into neurite which has the longer than cell body diameter. Especially, protrusion number was increased in Goα, Goiα, Gioα -F11 cell line at 36 hr (Fig. 3A). Through the quantitative analysis table, we can easily know the fact that the average of protrusion number was increased in Goα (4.2±0.7), Goiα (4.2±0.7) and Gioα(4.2±0.9)-F11 cell that can appear to interaction with Goα and PKA-Cα through the GTPase domain at the stage 36 hr. Interestingly, protrusion number is still increased until 48hr. In conclusion, GTPase domain of Goα is required for interaction with PKA-Cα. And the Goα constructs which has the ability to binds to PKA-Cα contributes to an increase in the number of protrusion at 36 hr. Besides, it is a noteworthy fact that dynamic changes of the protrusion formation occurred between 24 hr and 36 hr. Thus, we

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could observe the considerable difference in number of protrusion at 36 hr.

These data suggest that the interaction between Goα and PKA-Cα plays key role of protrusion formation in F11 cell stable cell line during differentiation. Moreover, in the process of neurite generation, the protrusion number of Goα, Goiα, Gioα-F11 cell was different as compared with normal F11 cell after at 36 hr (Fig. 4A). In order to find the mechanism about the effect of Goα in neurite generation of F11 cell, we compared with general cell polarization process. Each stage of cell polarization corresponds to differentiation time of F11 cell. If morphological change of F11 cell was regulated by Goα protein during differentiation, Goα protein or PKA-Cα will affect a variety of signaling molecules which involved in each stage of cell polarization.

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Fig. 9. The extending protrusions of F11 cell that expresses GTPase domain is increased at 36hr. (A) F11 cell lines were induced to differentiate with forskolin 30 μM from 0 to 48 hr. Comparing with each cells, all types of F11 cell are similar each other up to 24 hr (stage 2). But, the number of protrusion in F11 cell line expressing Goα, Goiα and Gioα is increased at 36 hr (stage 3). The morphology of F11 cell expressing Giα is similar to the normal F11cell as bipolar formation. Scale bar 50 μM. Scheme model of general cell polarization shows that the cell morphology is different at each cell polarization stage. Each stage is parallel to differentiation times of F11 cell. (B) Quantitative analysis table of protrusion number per cell. For analysis, all of the protrusions which were extended from cell body are selected. The protrusion was determined as all extending processes from cell body, individually.

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IV. DISCUSSION

Since the existence of Goα protein has been well-known from various studies, many researchers have been tried to identify the function of Goα. But, the precise function of Goα has been elusived. We also have been interested in finding the functional role of the Goα

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