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 GoαWT and GoαQ205L 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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2
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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 (2nd Alexa 488 green). The anti-FLAG (2nd 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 protein. In early study, the previous evidence implies that Goα modulates cAMP-induced neurite outgrowth of F11 cell. Overexpression of GoαWT and GoαQ205L (does not bind to βγ complex; can’t return to inactive state) reduced neurite length and increased the number of neurite per F11 cell that was differentiated by 0.5 mM db-cAMP for 4 days. In order to identify influence of Goα in cAMP-triggered PKA signaling pathway, CREB activity which is one of the downstream molecules of PKA was measured in condition of overexpressing GoαWT and GoαQ205L. The result reveals that CREB activity was inhibited in GoαWT and GoαQ205L expressing F11 cell (Ghil et al., 2000). Therefore, the result has been shown the evidence that Goα can regulate the cAMP-triggered signaling pathway. Another supporting study demonstrates that Goα is important regulator of cAMP-triggered PKA signaling pathway and is involved in many neuronal development process of cell maturation (Strittmatter et al., 1994).
In this study, to investigate the function and role of Goα, we performed binding assay and F11 neuroblastoma cell differentiation study. As a result, Goα directly binds to PKA-Cα through GTPase domain of Goα. In a view of interaction, the additional experiments are necessary to find the specific binding site of GTPase domain of Goα. Probably, a few specific binding sites may exist in G1 part of GTPase domain of Goα. We investigated if
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GTPase domain of Goα is required for an increase in neurite number of F11 cell. The previous studies report that Go constitutes 5~10% of membrane protein in the neuronal growth cone. (Strathman et al., 1990, Tsukamoto et al., 1991) And Gi/o signaling pathway was required for proper central nervous system cell development that involved in neurite formation and growth (Bromberg et al., 2011). According to our result, the number of neurite which has the longer length than cell body diameter was increased in F11 cell overexpressing Goα. In the stable cell line, many protrusions were generated in F11 cell lines which expresses GTPase domain of Goα at 36 hr (stage 3). Protrusion was determined as all extending things from cell body regardless of cell body diameter. Especially, we mentioned the protrusion concept instead of neurite in the stable F11 cell line. In stable cell line, the sustaining expression of Goα protein seems to affect the generation of protrusion depending on differentiation time. The dynamic changes of cell morphology seem to be emerged at transition period between 24 hr (stage 2) and 36 hr (stage 3).
Development stage of neuronal polarization can be divided into five stages. At stage 1, several short and thin filopodia were generated. Subsequently, the matured protrusions from filopodia can be grown up into immature neurites at stage 2. After stage 2, immature neurites begin to grow up, rapidly. Interestingly, selected one neurite becomes longer than the other neurites at the stage 3. Finally, the non-selected other neurites become dendrites at the stage 4 (Nariko et al., 2007). When we compare general cell polarization and F11 cell differentiation stage, each differentiation times correspond to the stage of cell polarization.
Therefore, Goα protein may affect cell polarization related the intracellular molecules in transition period between 24 hr (stage 2) and 36 hr (stage 3).
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In finding mechanism of Goα in F11 cell, recent study reveals direct evidence that the interaction of Goα and PKA-Cα through GTPase domain of Goα blocks translocation of PKA-Cα into nucleus in cos7 cells with FLAG-tagged Goα and Gioα constructs. Therefore, Goα regulates PKA-triggered signaling pathway by inhibition of translocation PKA-Cα into nucleus. Generally, catalytic subunits of PKA were observed in both cytoplasm and nucleus.
But, PKA-Cα was distributed in only cytoplasm of Goα and Gioα expressing cos7 cell but not Giα, Goiα (data not shown, but the figure locates in Ghil et al., 2006).
Our result shows that Goiα(GSTGoαΔG2) can bind to PKA-Cα through G1 domain. Thus, the translocation of PKA-Cα into nucleus should be inhibited by Goiα. We suppose that the function of two proteins may be differently exhibited between transfected cos7 cell and F11 stable cell line. To confirm the hypothesis, it is necessary to check the distribution of PKA-Cα in F11 stable cell line.
Taken together, physiological phenomena such as a difference of neurite and protrusion number of F11 cell might be affected by the interaction with Goα and PKA-Cα through the GTPase domain of Goα. These roles will affect to differentiation and maturation of F11 cell.
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V. CONCLUSION
This study demonstrates that GTPase domain of Goα is essential for interaction between Goα and PKA-Cα. Goα can modulate PKA-triggered signaling pathway by interaction with PKA-Cα, directly. Physiologically, the number of neurite was increased by interaction of Goα and PKA-Cα through GTPase domain of Goα in forskolin-induced differentiated F11 neuroblastoma cell for 2 days. Furthermore, each of Goα, Goiα and Gioα stable cell line which were formed the interaction with PKA-Cα except for Giα protein has many protrusions after 36 hr. Therefore, the interaction between Goα and PKA-Cα through the GTPase domain affects the neurite generation in the differentiation process of F11 cell. Our results suggest that the function of Goα is required for cell maturation process.
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REFERENCES
1. Bromberg KD, lyenger Ravi, Cijiang He John: Regulation of neurite outgrowth by Go/i signaling pathways. Front Biosci, 13: 4544–4557, 2011
2. Ghil S, Choi JM, Kim SS, Lee YD, Liao Y, Birnbaumer L, Suh-Kim H: the Compartmentalization of protein kinase A signaling by the heterotrimeric G protein Go.
Proc Natl Acad Sci USA, 103:19158–19163, 2006
3. Ghil SH, Kim BJ, Lee YD, Suh-Kim H: Neurite Outgrowth Induced by Cyclic AMP Can Be Modulated by a Subunit of Go. J Neurochem, 74:151–158, 2000
4. Hepler JR, Gilman AG: G proteins. Trends Biochem Sci, 17:383-387, 1992
5. Jiang M, Spicher K, Boulay G, WangY, Birnbaumer L: Most central nervous system D2 dopamine receptors are coupled to their effectors by Go. Proc Natl Acad Sci USA, 98(6):3577-2582, 2001
6. Jordan JD, He JC, Eungdamrong NJ, Gomes I, Ali W, Nguyen T, Bivona TG, Philips MR, Devi LA, Iyengar R: Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G (alpha)o/i-triggered proteasomal degradation of Rap1GAPII. J Biol Chem 280:11413-11421, 2005
7. Jung Hee Won, Jung Sik Park, Hyun Hee Ju, Soyeon Kim, Haeyoung Suh-Kim, Sung Ho Ghil: The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions. Cell Signal, 20(5):884–91, 2008
8. Law SF, Yasuda K, Bell GI, Reisine T: Gi alpha 3 and Go alpha selectively associate with the cloned somatostatin receptor subtype SSTR2. J Biol Chem, 268(15):10721-10727, 1993
- 38 -
9. Minaba M, Ichiyama S, Kojima K, Ozaki M, Kato Y: Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals. FEBS J, 273(24):5508-5516, 2006
10. Morishita R, Kato K, Asano T: GABAB receptors couple to G proteins Go, Go* and Gi1 but not to Gi2. FEBS lett, 271(1-2):231-235, 1990
11. Mullaney I, Milligan G: Elevated levels of the guanine nucleotide binding protein, Go,
11. Mullaney I, Milligan G: Elevated levels of the guanine nucleotide binding protein, Go,