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Originally published In Press as doi:10.1074/jbc.M004069200 on August 30, 2000

J. Biol. Chem., Vol. 275, Issue 47, 36885-36891, November 24, 2000
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Nadrin, a Novel Neuron-specific GTPase-activating Protein Involved in Regulated Exocytosis*

Ayako HaradaDagger , Birei FurutaDagger §, Ken-ichi TakeuchiDagger , Makoto Itakura, Masami Takahashi, and Masato UmedaDagger ||

From the Dagger  Department of Molecular Biodynamics, The Tokyo Metropolitan Institute of Medical Science (RINSHOKEN), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, § Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo, 112-8610, Japan, and  Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan

Received for publication, May 12, 2000, and in revised form, August 30, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca2+-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca2+-dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Neurotransmitter release, a fundamental step in the process of synaptic transmission, is accomplished by the rapid membrane fusion of neurotransmitter-filled synaptic vesicles with the target plasma membrane (1, 2). The actin cytoskeleton has been proposed to play a number of roles in regulated exocytosis, particularly in endocrine and neural cells (3, 4). Morphological studies on chromaffin and neural cells demonstrated that most of the secretory vesicles are positioned at a distance of ~250 nm from the plasma membranes, suggesting the presence of a physical barriers to the movement of secretary vesicles toward the release site on plasma membranes (5-7). Localization of actin filaments using anti-actin antibodies or fluorescence-labeled phalloidin on the secretory cells has shown the presence of actin filament networks underneath the presynaptic plasma membranes and disassembly of the cortical actin filament network upon activation of the secretory cells (7-10). It has been proposed that the cortical actin filament networks act as a reservoir of vesicles ready for docking at the release sites and as a cortical barrier that must be reorganized to enable docking and fusion of synaptic vesicles with plasma membranes. Although several actin-depolymerizing proteins that mediate the actin filament disassembly, such as scinderin and gelsolin, are proposed to regulate filament disassembly and exocytosis (11-13), the molecular mechanisms underlying the precise regulation of actin filament networks during neurotransmitter release remain unknown.

The Ras superfamily of small GTP-binding proteins are molecular switches that regulate numerous cellular functions by controlling intracellular signaling events (14). The Rho family of GTP-binding proteins, consisting of Rho, Rac, and Cdc42 proteins, regulate a number of cellular functions that require the reorganization of actin-based structures (15, 16). Recent investigations lead to the idea that Rho family proteins are also involved in signaling pathways that control actin filament reorganization during exocytosis. In chromaffin cells, RhoA is specifically associated with the membrane of secretory chromaffin granules and is suggested to control the priming of exocytosis by modifying the cortical actin network (17, 18). In mast cells, activation of GTP-binding proteins by GTPgamma S1 induces reorganization of actin filaments (19). The expression of constitutively active mutants of either RhoA, Rac, Cdc42 proteins enhanced regulated exocytosis, and inhibition of endogenous Rac and Rho activities reduces the secretory response (20-22). These observations suggest that Rac and Rho are components of the signaling pathways that lead to the cytoskeleton reorganization necessary for exocytosis.

The GTP-binding proteins cycle between an active GTP-bound form and an inactive GDP-bound form. This process is regulated by three types of factors: guanine nucleotide exchange factors, which stimulate the interconversion of the GDP-bound inactive form to GTP-bound active form; guanine nucleotide dissociation inhibitors, which inhibit this reaction; and GTPase-activating proteins (GAPs), which stimulate conversion from the GTP-bound form to GDP-bound form (14, 23). GAPs for Rho family of GTP-binding proteins belong to a family whose members share significant sequence homology in a conserved GAP domain, the RhoGAP domain (14). More than 15 proteins containing RhoGAP domains have been identified in mammalian cells, and these proteins are suggested to serve as downstream effectors in signal transduction events as well as controlling the activities of Rho family proteins (14, 24, 25). Among these regulators of GTP-binding proteins, Rho guanine nucleotide dissociation inhibitors was shown to be involved in regulated exocytosis (26), but the roles of GAPs or guanine nucleotide exchange factors in the process of exocytosis remain unknown.

We report here the identification of a novel neuron-associated developmentally regulated protein, Nadrin. Nadrin contains the conserved GAP domain that is active on the Rho family proteins, and it colocalized with the cortical actin filaments in PC12 cells. Expression of either the entire protein or various domains of Nadrin in PC12 cells cotransfected with growth hormone strongly affects high K+-induced growth hormone secretion. The expression of Nadrin is restricted to neuronal cells and is highly correlated with maturation of the central nervous system. We propose that Nadrin plays an important role in controlling neurotransmitter release by regulating reorganization of the cortical actin filament network in nerve endings.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cloning of Nadrin cDNA-- A lambda gt11 cDNA library constructed from the brain of an 8-wk-old female Wistar rat was screened by using monoclonal antibody 3A10, as described previously (27). Positive clones were purified by successive rounds of plaque purification. cDNA inserts were subcloned into a pBluescript vector (Stratagene, La Jolla, CA). The library was rescreened with a partial cDNA probe of Nadrin from clone N (Ref. 27; GenBankTM accession number AF022966) by DNA hybridization. Positive clones were purified, and cDNA inserts were subcloned into a pBluescript vector. The samples were sequenced on an ALFred DNA sequencer (Amersham Pharmacia Biotech) using an AutoCycle sequencing kit (Amersham Pharmacia Biotech). Sequence analyses were performed using Genetix Version 10.0 (Software Development Co. Ltd., Tokyo, Japan), and data base searches were performed using BLAST (28) and FASTA programs (29). The Nadrin amino acid sequence was analyzed using the COILS version 2.2 (30) and the PEST find programs (31).

GAP Assay-- An expression vector for the GAP domain of Nadrin fused to glutathione S-transferase (GST) was made by subcloning the cDNA fragment encoding amino acid residues 218-482 of Nadrin into a pGEX-vector (Amersham Pharmacia Biotech). An expression vector for the GAP domain of 3BP-1 (amino acids 184-420) (32) was made similarly. GST fusion proteins were expressed in DH5alpha Escherichia coli cells, purified by affinity chromatography on glutathione-Sepharose (Amersham Pharmacia Biotech) with elution buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 10 mM glutathione), and dialyzed into 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol. The GAP activity of Nadrin was assayed using a GTPase-activating protein assay biochem kit (Cytoskeleton, Denver, CO) according to the manufacturer's protocol. Briefly, recombinant GST-tagged RhoA, Rac1, and Cdc42 (1.5 µM) were preloaded 10 min at 30 °C with 10 µCi of [gamma -32P]GTP (6000 Ci/mmol, NEN Life Science Products) in 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.4 mM dithiothreitol, and 8.3 mM NaCl. After the addition of 25 mM MgCl2, preloaded GTPases (final concentration, 100 nM) were diluted in buffer (10 mM Tris-HCl (pH 7.5), 0.05 mM dithiothreitol, 0.5 mg/ml BSA, 0.5 mM GTP), and proteins (20, 100, or 200 nM GST-GAP fusion protein) were added to reaction mixture. Aliquots were incubated for min at 25 °C, and the reaction was stopped by adding 1 ml of ice-cold buffer (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2) and affinity beads and incubated for 15 min at 4 °C. The beads were washed with ice-cold buffer and subjected to scintillation counting.

Antibodies-- An anti-Nadrin antibody was raised in Japanese White female rabbits against the synthetic peptide CPPKPGNPPPGHPGGQSSPG, corresponding to amino acids 562-580 of Nadrin, with the addition of an extra cysteine to the N-terminal end, as described previously (33). Briefly, the peptide was conjugated with maleimide-activated keyhole lympet hemocyanin (Pierce) according to the manufacturer's protocol. Two rabbits were immunized subcutaneously with 250 µg of synthetic peptide keyhole lympet hemocyanin conjugate in Freund's complete adjuvant (Difco Laboratories, Detroit, MI), followed by three injections of the antigen in Freund's incomplete adjuvant (Difco) at 4-week intervals. Immunogloblin G (IgG) was isolated from the pre-immune and immune sera of the rabbits by ammonium sulfate precipitation and affinity chromatography on a protein G-Sepharose column (Amersham Pharmacia Biotech). Monoclonal antibody 1D12 was used for synaptotagmin detection (34).

Immunoblotting Analysis-- Organs were isolated from Wistar rats, then homogenized in 10 volumes (w/v) SET buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, 5 mM N-ethylmaleimide) containing 1 mM phenylmethanesulfonyl fluoride at 4 °C. Protein levels in the homogenates were determined using the BCA system (Pierce). The protein concentrations of the samples were adjusted, and the samples were placed in reducing sample buffer. Immunoblotting was performed as described previously with some modifications (27). Proteins from various sources were separated by SDS-polyacrylamide gel electrophoresis in 7.5% (w/v) polyacrylamide gels and blotted onto nitrocellulose membranes 4 °C using a protein transfer system (ATTO Co., Tokyo, Japan). The membranes were blocked by incubation in blocking buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5% skim milk (Difco)) for 1 h at room temperature with gentle shaking and then incubated with anti-Nadrin antibody for 2 h at room temperature (IgG fraction, 4 µg/ml) in a blocking buffer containing 0.2% Tween 20. Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech) (1/4000 dilution with the blocking buffer containing 0.05% Tween 20) using ECL Western blotting detection reagents (Amersham Pharmacia Biotech).

Cell Culture-- Subcloned PC12 cells (kindly provided by Dr. Y. Fukui, The University of Tokyo) were grown in Dulbecco's modified minimum essential medium (Asahi Techno Glass Co., Tokyo, Japan) with 10% horse serum (Life Technologies, Inc.) and 5% fetal bovine serum (JRH Biosciences, Lenexa, KS). NIH3T3 cells were grown in Dulbecco's modified minimum essential medium with 5% calf serum (JRH Biosciences). Neuronal and glial cell cultures were performed as described previously (27).

Construction of Expression Vectors-- The cDNA constructs encoding the C-terminal GFP-tagged variants of Nadrin (amino acids 1-780), domain I + II (amino acids 1-465), domain I (amino acids 1-233), and domain III (amino acids 463-780), were generated by PCR using primers that engineered 5' XhoI and 3' BamHI restriction sites into XhoI- and BamHI-digested pEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA). To create a Nadrin mutant that lacks GAP activity, a point mutation was introduced so as to alter Arg-288, which is suggested to be required for the catalytic activity of GAP (35, 36) to Ala by overlap PCR (37). The PCR products were digested with XhoI and BamHI and ligated into XhoI- and BamHI-digested pEGFP-N1 (CLONTECH Laboratories). The nucleotide sequences of PCR products were confirmed by DNA sequencing of both strands.

Transfection and Immunofluorescence Microscopy-- PC12 cells were plated at a density of 7 × 105 cells on 35-mm dishes and grown overnight. The cells were transfected with 2 µg of Nadrin expression vector by using LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.). After 1 day, transfected cells were seeded onto glass coverslips coated with poly-L-lysine (100 µg/ml, Sigma) and then cultured for 1 day or treated for 2 days with 50 ng/ml nerve growth factor (Chemicon International Inc., Temecula, CA) to induce cell differentiation. Immunocytochemistry was performed as described previously (38). Briefly, the cells were fixed with 3.7% formaldehyde for 15 min and washed three times with PBS and blocked PBS containing 2% BSA (2% BSA-PBS) for 30 min at room temperature. For detection of synaptotagmin, the fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 4 min. After being washed with PBS, the cells were incubated with monoclonal antibody 1D12 (25 µg/ml) in 2% BSA-PBS for 16 h at 4 °C. The cells were then washed with PBS and incubated with Cy3TM-conjugated goat-anti mouse IgG (Amersham Pharmacia Biotech) diluted 1/400 with 2% BSA-PBS for 1 h at room temperature. For actin filament staining, fixed cells were incubated with tetramethylrhodamine B isothiocyanate-labeled phalloidin (Sigma) (1/200 dilution PBS) for 45 min at room temperature. The cells were washed five times in PBS and mounted on microscope slides for observation and photography. The cells were examined with a confocal imaging system (LSM510; Carl Zeiss, Oberkochen, Germany). NIH3T3 cells were plated at a density of 6 × 104 cells/well in 24-well dishes and grown overnight. The cells were transfected with 2 µg of GFP-Nadrin expression vector using TransFast reagent according to the manufacturer's instructions (Promega). After 1 day, transfected cells were seeded onto glass coverslips and cultured for 1 day. Immunocytochemistry was performed as described above. Immunofluorescence microscopy was carried out using an Axiovert 135(Carl Zeiss).

Transfection and Growth Hormone Secretion Assay-- PC12 cells were plated at a density of 9 × 105 cells on collagen-coated 35-mm dishes (Vitrogen 100, Collagen Corporation, Palo Alto, CA) and incubated for 16-20 h. The cells were then co-transfected with 1.5 µg of pSIphi GH (39) and 1.5 µg of the indicated expression vector using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. GH release experiments were performed 72 h after transfection. PC12 cells were washed with Tyrode's-HEPES (20 mM HEPES (pH 7.4), 137 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2) and incubation for 4 min with low K+ buffer (Tyrode's-HEPES containing 137 mM NaCl, 5 mM KCl) or high K+ buffer (Tyrode's-HEPES containing 87 mM NaCl, 55 mM KCl). The amounts of GH released into the medium and retained in the cells were measured using a radioimmunoassay kit (Nichols institute, San Juan Capistrano, CA) or an immuno enzymometric assay kit (Tosoh Co., Tokyo, Japan). Secretion was expressed as a percentage of GH amounts released into medium relative to the total cellular GH amounts (40).

Subcellular Fractionation-- Cultures of PC12 cells (2-3 × 107 cells) were placed on ice and rinsed and resuspended in SET buffer containing 1 mM phenylmethanesulfonyl fluoride and a protease inhibitor mixture (CompleteTM, Life Technologies, Inc.). Cells were homogenized with 20 strokes of a Teflon glass homogenizer and then centrifuged for 30 min at 100,000 × g. The supernatant was saved (cytosol), and the pellet was homogenized with a Teflon glass homogenizer in one of the extraction buffers. The suspension was then incubated for 30 min at 4 °C and centrifuged for 30 min at 100,000 × g. The supernatant and pellet were saved and dissolved in sodium dodecyl sulfate sample buffer. The extraction buffers were SET buffer containing the inhibitors mentioned above and one of 0.5% Triton X-100, 0.5% deoxycholate, or 0.5 M KCl.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cloning of Nadrin with GAP Activity on the Rho Family Proteins-- In a previous study we showed that monoclonal antibody 3A10 recognizes a series of developmentally expressed brain proteins with molecular masses of 150-, 120-, 118-, 106-, 104-, 79-, and 77-kDa, whose expression is correlated well with the maturation of the central nervous system (27). We purified the 79- and 77-kDa 3A10 antigens and identified them as synapsin Ia and Ib, respectively, by analysis of the sequences of peptide fragments (27). In this study, we cloned a gene encoding a 3A10-reactive 104-kDa protein by immunological screening of expression libraries constructed from adult rat brains. We designated the protein encoded by the gene as Nadrin (neuron-associated developmentally regulated protein) because its expression is neuron-specific and developmentally regulated (see below).

As shown in Fig. 1A, Nadrin cDNA encodes a 780-amino acid protein composed of three distinct domains that are schematically indicated in Fig. 1B. Primary sequence alignments indicated that domains I and II of Nadrin (amino acids 1-458) share 51% identity to mouse 3BP-1 (amino acids 1-406) (32) and 62% identity to the human KIAA0672 protein (amino acids 1-470) (41). Domain I (amino acids 1-248) is 24% identical to the N terminus of the endophilin II, which is predicted to form a coiled-coil structure (42). Indeed, amino acids 165-200 of Nadrin are predicted by the COILS algorithm (30) to have a nearly absolute probability of forming a coiled-coil structure. Other regions within domain I were also predicted to possess coiled-coil-forming motifs with lower probability than amino acids 165-200. Domain II (amino acids 249-458) showed an extensive homology to GAPs of the Rho family GTPases (RhoGAPs) (24, 43). Fig. 1C compares the RhoGAP domain of Nadrin to the RhoGAP domains of 3BP-1 (32), ABR (44), beta -chimaerin (45), Myr 5 (46), p190 (47), and p50rhoGAP (48). Domain III (amino acids 498-780) comprises a serine/threonine/proline-rich region in which the proline, serine, and threonine content is approximately 50%. In addition, domain III contains one region of 29 successive glutamines (amino acids 596-624), one PEST sequence (amino acids 653-679) (31), two nuclear localization signals (amino acids 479-481, 552-557) (49), and one potential SH3 binding motif (amino acids 704-710) (50).


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  		Fig. 1.   The predicted amino acid sequence of Nadrin. A, the region similar to GAPs of Rho family GTPases (RhoGAPs) is boxed. The predicted coiled-coil structure is underlined, and a PEST sequence is double-underlined. B, schematic diagram illustrating the domain structure of Nadrin. Nadrin is composed of three distinct domains; domain I (amino acids 1-248), domain II (amino acids 249-458), and domain III (amino acids 498-780). Domain I contains the predicted coiled-coil structure, and domain II is composed of a RhoGAP domain. The overall amino acid sequences of domain I and II show 51 and 62% identity, respectively, to mouse 3BP-1 and human KIAA0672 protein. Domain III is the serine/threonine/proline-rich region that contains 29 successive glutamines (amino acids 596-624) and an SH3 binding motif consensus sequence (amino acids 704-710). C, alignment of the RhoGAP domain of Nadrin with other RhoGAP family members that have known GAP activity.

The sequence analyses suggest that Nadrin functions as a GAP for members of Rho-family small GTP-binding proteins. To examine the GAP activity of Nadrin on proteins of Rho family, the GAP domain (amino acids 218-482) of Nadrin was produced as a GST fusion protein in E. coli and was assayed for its ability to activate the intrinsic GTPase activity of GTP-bound RhoA, Rac1, and Cdc42. As shown in Fig. 2A, the GAP domain was able to stimulate the intrinsic GTPase activity of RhoA, Cdc42, and Rac1. In a parallel analysis, the GAP domain of 3BP-1 stimulated the GTPase activity of Rac1 and Cdc42 but not that of RhoA (data not shown), which is consistent with previously published data (32). Overexpression of Nadrin in NIH3T3 cells markedly reduced the number of actin stress fibers and formed the ruffled membranes (Fig. 2B). These results clearly demonstrate that Nadrin functions as a GAP for Rho family proteins and as a regulator of cellular actin filament organization.


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  		Fig. 2.   GAP activity of Nadrin on Rho family GTPases. A, the intrinsic GTPase activities of RhoA, Rac1, and Cdc42 protein was measured in the presence and absence of various amounts of recombinant Nadrin (amino acids 218-482) containing the GAP domain. gamma Pi associated with the GTPases (100 nM) was determined at the 5-min time point in the absence or presence of the recombinant Nadrin proteins. B, effect of Nadrin on actin filament organization. NIH3T3 cells expressing GFP-tagged Nadrin were fixed and stained with tetramethylrhodamine B isothiocyanate-labeled phalloidin. Fluorescence micrographs compare GFP and phalloidin staining. Scale bar = 10 µm.

Developmentally Regulated Expression of Nadrin in Rat Brain-- Tissue distribution of Nadrin in adult rats was analyzed by using a rabbit polyclonal antibody raised against a synthetic peptide composed of amino acids 562-580. The anti-Nadrin antibody bound specifically to a 104-kDa protein when Nadrin was expressed in COS-7 cells (data not shown). In adult rat tissues, the 104-kDa protein band was specifically detected in a brain homogenate but not in other tissues, indicating that Nadrin is specifically expressed in brain (Fig. 3A). The expression of Nadrin was dependent on the developmental stage of the brain; Nadrin became detectable at the second postnatal week in the cerebral cortex and hippocampus and at the third postnatal week in the cerebellum and olfactory bulb (Fig. 3B). The expression level was maximal during the third and fourth postnatal weeks and remained high during adulthood. To examine whether Nadrin is expressed in neuronal or glial cells, primary cultures of neuronal and glial cells were established from the cerebral cortex of E18 rat brain. The neuronal and glial cultures consisted of 80% neurons and more than 95% glia, based on the immunocytochemical criteria for the expression of neuron-specific enolase and glial fibrillary acidic protein (27) (data not shown). Nadrin became detectable on the 14th day of neural cell cultivation, and the level of Nadrin expression increased during the culture period in neuronal cells (Fig. 4A). The expression of Nadrin in neuronal cells correlated well with the expression of neuron-specific enorase and synapsin I (27) (data not shown). Nadrin was not detected in glial cells (Fig. 4A). These results clearly indicate that Nadrin is a neuron-specific protein, and its expression is closely correlated with neuronal differentiation.


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  		Fig. 3.   Brain-specific and developmentally regulated expression of Nadrin. A, tissue homogenates from adult rat were analyzed by immunoblotting with an anti-Nadrin antibody. A specific band of 104 kDa was detected in brain but not in other tissues. B, regional expression of Nadrin at various developmental stages of brain was analyzed by immunoblotting with an anti-Nadrin antibody. P, postnatal day; w, weeks after birth; CR, cerebral cortex; CL, cerebellum; HP, hippocampus; Olf, olfactory bulb. *, these bands represent nonspecific background of the horseradish peroxidase-conjugated anti-rabbit IgG antibody, which also appeared in the absence of the anti-Nadrin antibody.


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  		Fig. 4.   Neuron-specific expression of Nadrin. A, expression of Nadrin in cultured neuronal and glial cells was analyzed by immunoblotting with the anti-Nadrin antibody. B, co-localization of Nadrin with synaptotagmin at the neurite terminals. GFP-tagged Nadrin was expressed in differentiated PC12 cells. Distribution of the GFP-tagged Nadrin and synaptotagmin is shown by double-labeling in confocal images. Superimposed (merge) images demonstrate the overlapping distribution of these proteins at the neurite terminals. Scale bar = 20 µm.

During development of the central nervous system, an increase in the expression of various synaptic proteins, such as synaptotagmin I, synapsin I, Rab3A, and Rab guanine nucleotide dissociation inhibitors, correlated well with axon terminal differentiation and maturation of neuronal connectivity (51-54). These proteins function in the regulated exocytosis of synaptic vesicles (1). To investigate the potential role of Nadrin in exocytosis, a GFP-tagged variant of Nadrin (Nadrin-GFP) was expressed in PC12 cells, and its localization was compared with that of synaptotagmin I, which was detected by an anti-synaptotagmin monoclonal antibody 1D12 (34). Immunoblotting analysis showed that the anti-Nadrin antibody bound specifically to a 104-kDa band in PC12 cells, indicating that Nadrin is present in PC12 cells (data not shown). Fig. 4B shows a single confocal optical plane that illustrates the punctate appearance of Nadrin and synaptotagmin throughout the cytoplasm of the cell. An intense co-localization of Nadrin with synaptotagmin was observed in the neurite terminals. These data on the regional and developmental stage-specific expression of Nadrin suggest that Nadrin may have a function in regulating neurotransmitter release at the synaptic terminal.

Involvement of Nadrin in Ca2+-dependent Exocytosis-- To examine whether Nadrin functions in exocytosis, PC12 cells were co-transfected with human GH and Nadrin, and the high K+-induced secretion of co-expressed GH was determined. The expressed GH is known to be stored in dense core vesicles and to be released in response to high K+ in the presence of extracellular Ca2+ (39, 40, 55). The basal secretion of GH, expressed as a percentage of the amount of GH released into medium containing 4.7 mM K+ relative to total cellular GH, is not significantly different between Nadrin-transfected cells and control cells transfected only with GH. In this assay system, an average of 37 ± 3.3% GH was secreted upon high K+ stimulation and the GH secretion was slightly, but significantly, enhanced in Nadrin-transfected cells (Fig. 5A, a). The mutant comprised of domain I and domain II also enhanced K+-induced release of co-expressed GH, whereas the mutant comprised of domain I significantly inhibited the release (Fig. 5A, a). It has been shown that Arg residue, which is highly conserved in GAP domains, is required for their catalytic activity (35, 36). The equivalent residue in Nadrin is Arg-288 and replacement of Arg-288 with Ala in the GAP domain of Nadrin reduced the GAP activity to one-fifth the original activity (data not shown). We replaced Arg-288 of Nadrin with Ala (Nadrin R288A) and transfected the mutant Nadrin into PC12 cells. No significant alteration in the high K+-induced exocytosis was observed with Nadrin R288A-transfected cells, suggesting that GAP activity is required for the enhancement of exocytosis by Nadrin-expression (Fig. 5A, b).


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  		Fig. 5.   Involvement of Nadrin in Ca2+-dependent exocytosis. A, effect of Nadrin and Nadrin mutants on GH secretion from PC12 cells. PC12 cells were co-transfected with pSIphi GH, which encodes human GH, and pEGFP-N1, which contains the indicated cDNAs encoding Nadrin (amino acids 1-780), domain I (amino acids 1-233), domain I + II (amino acids 1-465) (a) and Nadrin R288A (a Nadrin mutant altered Arg-288 to Ala) (b). GH secretion was induced with a low K+ solution (5.6 mM KCl) or a high K+ solution (55 mM KCl) in the presence of extracellular Ca2+ (1 mM). The GH secretion is expressed as a percentage of total GH content. Data are representative of four independent measurements, and statistically significant differences (p < 0.01) are marked by asterisks. B, intracellular localization of Nadrin. PC12 cells expressing GFP-tagged Nadrin were fixed and stained with tetramethylrhodamine B isothiocyanate-labeled phalloidin. Distribution of the GFP-tagged Nadrin, F-actin, and the superimposed images is shown by double-labeling in confocal images. Arrows indicate the regions where Nadrin colocalizes with F-actin. Scale bar = 5 µm. C, subcellular fractionation of Nadrin. Homogenates of PC12 cells were centrifuged to separate the cytosol (S) and pellet (P). The resulting pellet was solubilized with either 0.5% Triton X-100 (Tx), 0.5 M KCl, or 0.5% deoxycholate (Doc), followed by centrifugation. The presence of Nadrin in each supernatant (S) and pellet (P) fraction was examined by immunoblotting using an anti-Nadrin antibody.

Since Nadrin is already present in PC12 cells and was not significantly overexpressed in PC12 cells in our assay conditions (data not shown), it is difficult to see the additive effect of the expressed Nadrin on exocytosis. In contrast, significant inhibitory effect was observed when the domain I (coiled-coil-rich domain) was expressed in PC12 cells. Several studies have shown that the coiled-coil domain plays a critical role in formation of a ternary core complex containing synaptobrevin, syntaxin, and SNAP25, which is central to the process of synaptic vesicle docking and fusion (56). It is possible that the expression of the coiled-coil-rich domain I of Nadrin may disturb the structural organization of the exocytosis complex, which inhibits the Ca2+-dependent release of GH.

Analyses of the subcellular localization of the GFP-tagged Nadrin in PC12 cells showed a punctate appearance of Nadrin throughout the cytoplasm. Comparison of the distribution of Nadrin and F-actin labeled by tetramethylrhodamine B isothiocyanate-labeled phalloidin showed that there are clearly several subplasmalemmal regions where Nadrin and actin filaments were colocalized (indicated by arrows in Fig. 5B). To test biochemically whether the endogenous Nadrin was associated with the cytoskeletal components, cell lysates were incubated with Triton X-100 to solubilize membranes, and the Triton-insoluble cytoskeleton was pelleted by centrifugation (57, 58). Nadrin was predominantly present in the 100,000 × g-precipitated fraction, and the Triton-treatment did not release endogenous Nadrin into the supernatant, whereas 0.5 M KCl treatment readily released Nadrin (Fig. 5C). These results suggest that Nadrin was associated with cytoskeletal components in PC12 cells. We did not observe a significant change in the distribution of actin filaments after the expression of Nadrin in PC12 cells, although Nadrin regulates actin filament reorganization when expressed in NIH3T3 cells (Fig. 2B). Based on these observations, we proposed that Nadrin is involved in Ca2+-depenent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing reorganization of the cortical actin filaments.

The precise mechanisms by which Nadrin exerts its function in vivo remain to be elucidated. The C-terminal serine/threonine/proline-rich domain III of Nadrin contains a polyglutamine repeat and two nuclear localization signals. Recent studies (59, 60) show that a number of inherited neurodegenerative diseases are characterized by expanded polyglutamine repeats within the coding sequence of the disease gene. To begin to examine the possible relationship of Nadrin with these diseases, human cDNA showing a high similarity to Nadrin was amplified by reverse transcription-PCR, and the deduced amino acid sequence was compared with that of rat Nadrin. The polyglutamine repeat was deleted from the human Nadrin cDNA, although other coding regions showed extensive homology between rat and human Nadrin (data not shown). Additional genetic studies, including chromosomal mapping and mutational analyses of Nadrin, are now under way to clarify the involvement of Nadrin in these diseases.

Concerning the functional role of the domain III of Nadrin, our recent experiments showed that the GFP-tagged domain III expressed in 3T3 fibroblasts localized specifically in the nucleus, which suggests a function for Nadrin in the nucleus (data not shown). The discovery of a novel brain-specific multifunctional protein may provide valuable information concerning the regulatory mechanisms of the actin cytoskeleton during exocytosis and may generate an interesting paradigm for possible cross-talk between synaptic terminals and the nucleus.

    ACKNOWLEDGEMENTS

We thank Dr. Yoshitaka Ono and Dr. Hideki Shibata (Kobe University) for their advice on measuring GAP activity. We also thank Dr. Donald M. Marcus (Baylor College of Medicine, Houston, Texas) for helpful comments during preparation of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB042827.

|| To whom correspondence should be addressed. Tel.: 81-3-3823-2101 (ext.5419); Fax: 81-3-3823-2130; E-mail: umeda@rinshoken.or.jp.

Published, JBC Papers in Press, August 30, 2000, DOI 10.1074/jbc.M004069200

    ABBREVIATIONS

The abbreviations used are: GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; BSA, bovine serum albumin; GAP, GTPase-activating protein; GFP, green fluorescent protein; GH, growth hormone; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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