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Interaction of Heterotrimeric Go Protein with Protein Kinase A

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Interaction of Heterotrimeric Go Protein with

Protein Kinase A

by

Yoon Sang Phil

Major in neuroscience

Department of Biomedical sciences

The Graduate School, Ajou University

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Interaction of Heterotrimeric Go Protein with

Protein Kinase A

by

Yoon Sang Phil

A Dissertation Submitted to the Graduate School of

Ajou University in Partial Fulfillment of the Requirements

for the Degree of

M.S. in Biomedical sciences

Supervised by

Haeyoung Suh-Kim, Ph.D.

Major in neuroscience

Department of Biomedical sciences

The Graduate School, Ajou University

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This certifies that the dissertation

of Yoon Sang Phil is approved.

SUPERVISORY COMMITTEE

Haeyoung Suh-Kim

Young-Don Lee

Sung-Soo Kim

The Graduate School, Ajou University

December, 19th, 2011

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-ABSTRACT -

Go Protein Regulates Neurite Outgrowth through Interaction with

Protein Kinase A

Heterotrimeric G proteins mediate signal transduction generated by numerous neurotransmitters and hormones. Among all G-proteins, Goα, a member of the Go/i family, is the most abundant G protein in brain. Most functions of Goα are mediated by the Gβγ dimer;

effector(s) for its α-subunit have not been clearly defined. Previous study have shown that overexpression of Goα interfered cAMP signaling in a neuroblastoma cell line. This report investigated which subdomain of Goα mediates interaction with cAMP-dependent protein kinase (PKA). GST pull-down and co-immunoprecipitation assays revealed that the Goα::PKA interaction depends on the GTPase subdomain of Goα. F11 neuroblastoma cell line was used to carry out study about the physiological relevance of this interaction using various Goα mutants. When F11 cells were induced to differentiate with 30 μM forskolin, the neurite and protrusion number of F11 cell were increased in F11 cells overexpressing Goα protein that binds to PKA-Cα through the GTPase domain of Goα.

Key words: Goα Protein, GTPase domain, cAMP-dependent protein kinase (PKA), F11 cell

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TABLE OF CONTENTS

ABSTRACT ··· TABLE OF CONTENTS ··· LIST OF FIGURES ··· ⅳ . INTRODUCTION 1. Heterotrimeric G protein ··· 5

2. The α subunit of Go protein ··· 6

3. cAMP-dependent protein kinase (PKA) ··· 7

4. F11 neuroblastoma cell ··· 8

5. Purpose ··· 9

. MATERIALS AND METHODS 1. Cells ··· 10

2. Plasmids ··· 10

3. Manufacture of GST-fusion protein ··· 11

4. Establishment of stable F11-Goα, Giα, Goiα, Gioα cell line ··· 15

5. Cell culture ··· 15

6. Differentiation ··· 15

7. Quantification of the number of neurite and protrusion ··· 16

8. Co-immunoprecipitation ··· 16

9. GST pull down assay ··· 17

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11. Immunoblotting ··· 18 .

RESULTS

1. Goα protein directly interacts with PKA-Cα through the GTPase domain of Goα ··· 20 2. The interaction of Goα and PKA-Cα has an influence on neurite formation of

F11 cell ··· 25 3. The neurite formation was changed depending on differentiation time ··· 29

. DISCUSSION ··· 33 . CONCLUSION ··· 36 REFERENCES ··· 37 국문요약 ··· 41

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LIST OF FIGURES

Fig. 1. The general characteristic of heterotrimeric G protein ··· 6

Fig. 2. Standard model of PKA ··· 8

Fig. 3. Amino acid sequence anaylsis of Goα and Giα ··· 11

Fig. 4. GST-GoΔG2 ··· 13

Fig. 5. GST-GoΔNHLG1 ··· 14

Fig. 6. Goα protein directly binds to PKA-Cα, not Giα ··· 22

Fig. 7. Goα protein directly interacts with PKA-Cα through the GTPase domain of Goα ··· 24

Fig. 8. GTPase domain of Goα is essential for an increase in neurite number ··· 28

Fig. 9. The extending protrusions of F11 cell that expresses GTPase domain is increased at 36hr ··· 32

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I. INTRODUCTION

1. Heterotrimeric G protein

The heterotrimeric G protein was well-known to major upstream signal transducer in many intracellular signal transduction processes. Moreover, G protein coupled receptor (GPCR) that conjugated with various types of G protein was conformational changed by ligands such as neurotransmitters or hormones. Therefore, G protein mediates signal transduction from GPCR, a heptahelical seven transmembrane receptor in response from extracellular signals/stimuli (Pierce KL et al., 2002; Park PS et al., 2008). The G protein affects many intracellular effectors as ion channels, enzymes even small GTPase proteins. Especially, Gs (stimulatory) protein activates adenylyl cyclase from β-adrenergic receptor in response to β-adrenergic stimuli, whereas Gi (inhibitory) has opposite function that inhibits adenylyl cyclase (Hepler and Gilman et al., 1992). Heterotrimeric G proteins consist of three subunits, Gα (40kDa) and the tightly associated Gβγ (36kDa, 8kDa) subunits (Weng et al., 1998). The exchange from GDP to GTP at the Gα subunit causes dissociation from the βγ complex. Actived Gα subunit and βγ complex lead to stimulation other effectors such as ion channels, phospholiphase C (PLC), adenylyl cyclase (AD).

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Fig. 1. The general characteristic of heterotrimeric G protein. In inactive state, GDP (guanosine diphosphate) bound Gα protein binds to Gβγ complex. Inactive form of Gα was changed to active form which binds to GTP (guanosine triphosphate) by conformational changed GPCR. Dissociated Gα and βγ complex affect their effectors in intracellular. Each subunits get back together again.

2. The α subunit of Go protein

Among numerous GTP binding protein (G protein), especially, Goα protein was named “the other GTP-binding protein” is abundantly expressed in the brain and the growth cone membrane of neurite (Sternweis and Robishaw et al., 1984, Strittmatter et al., 1990). Goα protein was isolated from the bovine brain during purification of Giα protein for use as the substrate for PTX (Pertussis Toxin)-mediated ADP ribosylation (Neer et al., 1984). Goα protein is similar to Giα protein approximately 70%, the Goα protein was classified as a member of Gi/oα family. The α subunit of Go protein was actived by many types of GPCR including μ-and δ-opioid receptor (Ueda et al., 1988, Zhang et al., 2003), adenosine A1 receptor (Sweeney and Dolphin et al., 1995), dopamine D2 receptor (Jiang et al., 2001),

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GABAB receptor (Morishita et al., 1990), 5HT1p receptor (Wang et al., 1996),

SST(somatostatin) receptor 2 (Law et al., 1993), α2 adrenergic receptor (Nobles et al., 2005), muscarinic M2 and 4 receptors (Minaba et al., 2006, Wettschureck N et al., 2005). Actived GTP bound Goα protein affects some signal transduction molecules as second messenger in intracellular. And Goα protein turns back the inactive state again. Goα protein consists of three major domains: N-terminal domain, helical domain and GTPase domain which locates in direction of C-terminal and two linker domain referred to as linker1, 2 connects an interval of three major domains.

3. cAMP-dependent protein kinase (PKA)

PKA is one of the important kinases that phosphorylate many substrates in intracellular. PKA is regulated by cAMP, a second messenger that was synthesized by adenylyl cyclase from ATP (Adenosine Triphosphate). Among many G proteins, GS (stimulatory) protein

stimulates adenylyl cyclase which promotes PKA signaling pathway but Gi (inhibitory) does not. Under low concentration of cAMP, PKA exists as inactive state of heterotetramer consisting of two regulatory and two catalytic subunits. Ultimately, two catalytic subunits were diassociated from two regulatory subunits which bound to four cAMP. The actived free catalytic subunits phosphorylate a variety of intracellular signal molecules such as Rap-1 in cytoplasm. Futhermore, some free catalytic subunit of PKA translocate into nucleus to phosphorylates transcription factor such as cAMP response element binding protein (CREB).

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Fig. 2. Standard model of PKA. Unlike Gαi, actived Gαsprotein stimulates adenylyl cyclase which promotes PKA signaling pathway. The cAMP was generated by active adenylyl cyclase from ATP. The four cAMP bind to two regulatory subunits of PKA, leading to releasing free catalytic subunits of PKA.

4. F11 neuroblastoma cell

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.

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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 2×106 cell. Next

day, 293T cells were transiently transfected with FLAG-tagged Goα, Giα, Goiα, Gioα and PKA-Cα (pcDNA3-Cα)by CaPO4 method. 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).

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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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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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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

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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

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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

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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, are associated with differentiation of neuroblastoma x glioma hybrid cells. FEBS lett, 244(1):113-118, 1989

12. Nariko Arimura & Kozo Kaibuchi: neuronal polarity: from extracellular signals to intracellular mechanisms. Nature Reviews Neurosci 8: 194–205, 2007

13. Neer, EJ, Lok J M, and Wolf, LG: Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase). J Biol Chem, 259:14222–14229, 1984

14. Nobles M, Benians A, Tinker A: Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc Natl Acad Sci USA, 102(51):18706-18711, 2005 15. Park PS, Lodowski DT, Palczewski K: Activation of G protein-coupled receptors:

beyond two-state models and tertiary conformational changes. Annu Rev Pharmacol Toxicol, 48:107–1419, 2008

16. Pierce, KL: Seven-transmembrane receptors. Nat.Rev. Mol. Cell Biol, 3:639–650, 2002 17. P.C. Sternweis, JD, Robishaw, J: Isolation of two proteins with high affinity for

guanine nucleotides from membranes of bovine brain. Biol. Chem, 259:13806, 1984 18. Strathmann M, Wilkie TM, Simon MI: Alternative splicing produces transcripts

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encoding two forms of the alpha subunit of GTP-binding protein Go. Proc Natl Acad Sci USA, 87(17): 6477–81, 1990

19. Strathmann M, Simon MI. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Proc Natl Acad Sci USA, 87(23):9113–7, 1990 20. Strittmatter SM, Fishman MC, Zhu XP: Activated mutants of the alpha subunit of G(o)

promote an increased number of neuritis per cell. J Neurosci, 14: 2327–2338, 1994 21. Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ, Fishman MC: Go is a major

growth cone protein subject to regulation by GAP-43. Nature, 344: 836–841, 1990 22. Sweeney MI, Dolphin AC: Adenosine A1 agonists and the Ca2+ channel agonist bay K

8644 produce a synergistic stimulation of the GTPase activity of Go in rat frontal cortical membranes. J neurochem, 64(5):2034-2042, 1995

23. Tsukamoto T, Toyama R, Itoh H, Kozasa T, Matsuoka M, Kaziro Y: Structure of the human gene and two rat cDNAs encoding the alpha chain of GTP-binding regulatory protein Go: two different mRNAs are generated by alternative splicing. Proc Natl Acad Sci USA, 88(8):2974–8, 1991

24. Ueda H, Harada H, Nozaki M, Katada T, Ui M, Satoh M, Takagi H: Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. Proc Natl Acad Sci USA, 85(18):7013-7017, 1988

25. Wang HY, Fiorica-Howells E, Pan H, Gershon MD, Friedman E Myenteric ganglionic 5-hydroxytryptamine (1P) signal transmission is mediated via Go protein. J Pharmacol Exp Ther, 277(1):518-24, 1996

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27. Wettschureck N, Offermanns S: Mammalian G proteins and their cell type specific functions. Physiol Rev, 85:1159-1204, 2005

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국문요약

-Goα 단백질과 cAMP 의존성 단백질 인산화 효소 A (PKA)의

결합에

따른 F11 세포의 신경돌기 형성 조절

아주대학교

의생명과학과 신경과학전공

윤 상 필

(지도교수: 서 해 영)

현재까지 뇌에 풍부하게 발현되는 Go protein 의 기능과 역할이 아직까지 제대로 밝혀진 바가 없어 많은 실험 연구의 대상이 되며 추측하건데 알려진 많은 생리학적 변화의 주된 원인으로 중요하게 작용할 수 있을 것으로 예상된다. 그리하여 본 연구에서 기존에 밝혀진 선행 연구를 토대로 세포 내 에 Go protein 특성을 찾아내고 실제 F11 과 같은 신경모세포종(neuroblastoma)에서 이 단백질의 기능과 역할을 관찰하여 이와 관련된 mechanism 을 밝히고자 하였다.

먼저, Go protein 은 Gi protein 과 매우 유사하여 같은 그룹에 속하지만 adenylyl cyclase 를 억제하는 Gi 와 달리 Go 는 그렇지 않다. 그럼에도 불구하고 뇌에 풍부하게 발현되는 단백질로 그 역할이 기대대는 바 선행 연구를 시작으로 처음으로 Goα 가 직접적으로 cAMP-dependent PKA signaling pathway 의 중요한 신호 분자인 PKA-Cα 와 결합함으로써 그와 관련된 신호 전달 체계를 조절할 수 있다는 것을 밝혀냈고 그와 더불어 PKA-Cα 와 결합하는 Go 의 결합부위 GTPase domain 을 찾아냈다. 이것을 증명하기 위해 Go 단백질의 α 소단위체의 여러 부위들이 없어진 construct 을 만들고 그것을 가지고 GST pull down assay 와 Co-immunoprecipitation 실험을 진행한 결과, Goα 의 GTPase domain 을 가진 construct 들 만이 PKA-Cα 와 결합하였고, 그렇지 않은 것들은 결합하지 않았다. 따라서 Go protein 과 PKA-Cα 와 결합하는데 있어서 Goα protein 의 GTPase

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domain 이 중요하다는 것을 알게 되었다. 결국, Goα protein 은 PKA 와 관련된 신호전달체계를 조절할 수 있다는 것을 의미하여, 실제로 이전에 발표된 논문에서 둘간의 결합이 PKA 의 free catalytic 소 단위체의 핵으로의 이동을 억제하는 것으로 확인 되었다. 그리하여 그 두 개의 단백질 간의 결합에 따른 생리학적 의미를 부여하기 위해서 PKA-Cα 와 결합하는데 필요한 Goα 단백질의 GTPase domain 결합부위를 가진 Goα protein 과 그렇지 않은 다양한 Goα protein 들을 과 발현시킨 F11 신경모세포종 세포를 cAMP 나 forskolin 으로 분화시켰다. 그리고 나서 세포가 가지는 neurites 의 개수를 직접 측정하여 그들간의 형태적인 차이점을 분석하고 그와 같은 변화 유도 기작을 밝혀내는 데 초점을 두었다.

지금까지의 결과들을 종합해 보면, Goα protein 이 PKA-Cα 와 결합하는데 있어서 Goα 의 GTPase domain 이 중요하다는 것을 알게 되었으며, 두 단백질간의 결합이 세포 내에서 PKA-Cα 의 핵 내로의 이동을 억제함으로써 신호 전달 체계를 조절하는 것으로 나타났다. 그와 같은 사실들의 생리학적인 의미를 부여하기 위해서 F11 세포에 Goα 를 과발현시켰을 때, neurite 의 개수가 일반적인 F11 cell 보다 증가하는 것으로 나타났다. 이는 아마도 두 단백질간의 결합에 따른 PKA-Cα 의 핵 내로의 억제로 인해 일어나는 것으로 보이며, 그 생리적인 변화 원인을 추측해 볼 수 있다. 아마도 Goα protein 이 PKA-Cα 와 결합하여 활성화된 PKA 의 소단위체의 핵 내로의 이동을 차단함에 따라 세포질에 구획화를 유도함으로써 실제 F11 cell 의 neurite 의 생성과 성장을 조절할 것으로 추측된다. 앞으로 이와 더불어 좀 더 구체적인 메커니즘을 밝혀내고 자 뉴런 세포의 성숙과정과 관련된 많은 단백질과의 관계를 파악하고 실제로 성숙단계에서 발생하는 다양한 생리학적 현상과 관련하여 추가적인 실험이 진행될 필요가 있다.

Key words: Goα 단백질, cAMP 의존성 단백질 인산화 효소, F11 신경모세포종, 신경돌기

수치

Fig.  1.  The  general  characteristic  of  heterotrimeric  G  protein.  In  inactive  state,  GDP  (guanosine diphosphate) bound Gα protein binds to Gβγ complex
Fig.  2.  Standard  model  of  PKA.  Unlike  Gαi,  actived  Gαs protein  stimulates  adenylyl  cyclase  which  promotes  PKA  signaling  pathway
Fig. 3. Amino acid sequence anaylsis of Goα and Giα. Goα shares 71% amino acid with  Giα
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
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