A Direct Inhibitory Role for the Rab3-specific Effector, Noc2, in Ca2+-regulated Exocytosis in Neuroendocrine Cells*

Lee P. HaynesDagger, Gareth J. O. Evans, Alan Morgan, and Robert D. Burgoyne§

From the Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom

Received for publication, August 2, 2000, and in revised form, December 4, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rab proteins comprise a family of GTPases, conserved from yeast to mammals, which are integral components of membrane trafficking pathways. Rab3A is a neural/neuroendocrine-specific member of the Rab family involved in Ca2+ -regulated exocytosis, where it functions in an inhibitory capacity controlling recruitment of secretory vesicles into a releasable pool at the plasma membrane. The effector by which Rab3A exerts its inhibitory effect is unclear as the Rab3A effectors Rabphilin and RIM have been excluded from for this role. One putative Rab3A effector in dense-core granule exocytosis is the cytosolic zinc finger protein, Noc2. We have established that overexpression of Noc2 in PC12 cells has a direct inhibitory effect upon Ca2+-triggered exocytosis in permeabilized cells. We demonstrate specific nucleotide-dependent binding of Noc2 to Rab3A and show that the inhibition of exocytosis is dependent upon this interaction since Rab3A binding-deficient mutants of Noc2 do not inhibit exocytosis. We propose that Noc2 may be a negative effector for Rab3A in regulated exocytosis of dense-core granules from endocrine cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The secretory pathway of eukaryotic cells is essential to the maintenance of cellular architecture and, in certain specialized cell types, serves as the basis for cell-cell communication. Intracellular transport occurs via the targeted passage of transport vesicles between distinct cellular compartments and is dependent upon the complex interplay of a large number of protein factors (1-4). Rab proteins comprise a large family of monomeric GTPases that are intimately linked to the secretory pathway in eukaryotes (5). Distinct Rabs appear to operate at different transport steps within the cell, exerting an essential or regulatory function on membrane fusion events (6) including in the mediation of vesicle tethering to target membranes. Rab3A is a mammalian, neural/endocrine-specific Rab protein present on synaptic vesicle and secretory granules (7-10) that appears to be involved in the control of Ca2+-regulated fusion of vesicles with the plasma membrane. Experiments using Rab3A knockout mice have demonstrated a nonessential role for this protein in regulating the efficiency of neurosecretion, possibly by limiting the recruitment of synaptic vesicles into the releasable pool at the plasma membrane for subsequent fusion events (11, 12). Rab3 mutants in Caenorhabditis elegans are also viable but have a reduction in synaptic vesicles close to the active zone (13). This suggests a role for Rab3 in vesicle tethering in common with other Rab proteins (14, 15). It is likely that Rab3 has multiple functions in exocytosis (16). Active forms of Rab3A inhibit exocytosis in various endocrine and neuroendocrine cell types (8, 17-19) and in neurons (20) consistent with an inhibitory function of Rab3A in regulated exocytosis.

The mechanism underlying the inhibitory effect of Rab3A on regulated exocytosis is unknown. It is likely that one or more specific Rab3A effector proteins that interact with the GTP-bound form of Rab3A are responsible for this inhibitory phenotype, and several such factors have now been identified and characterized. The first of these was Rabphilin3A (21), a 78-kDa neurally expressed protein that specifically binds to the GTP-bound state of Rab3A (22). The N-terminal portion of Rabphilin3A contains a zinc finger domain, while the C-terminal half of the protein harbors two C2 domains, which mediate Ca2+-dependent binding to phospholipids (23-25). In addition, Rabphilin3A also binds to alpha -actinin, a cytoskeletal protein involved in actin bundling, and hence may serve to integrate Rab3A activation with cytoskeletal rearrangements necessary for vesicle dynamics (26). Other Rab3A-specific effectors include RIM (27) and Noc2 (28). RIM is a large multidomain protein that localizes to pre-synaptic active zones and associates specifically with the GTP-bound form of Rab3A via a zinc finger motif. The function of RIM is as yet undetermined, although its limited homology to putative tethering proteins, along with its plasma membrane localization may link it to a pre-docking role via Rab3A (29). Overexpression of Rabphilin 3A (30) or an N-terminal fragment of RIM stimulates exocytosis (27). The use of mutagenesis and analysis of gene knockouts has ruled out a requirement for Rab3A interaction for these effects and have eliminated Rabphilin 3A or RIM as the inhibitory effectors of Rab3A (31-34). Another candidate Rab3A-interacting protein (35) that has been suggested to be involved in the inhibition of exocytosis (32) is calmodulin. This interpretation is problematic, however, as calmodulin has been shown to have a stimulatory effect on exocytosis (36). In addition, the binding of calmodulin to Rab3A is of low affinity and is not dependent on the nucleotide state of Rab3A (35). This is, therefore, not consistent with calmodulin acting as a Rab3A effector.

Noc2 (No C2 domain) is a 38-kDa cytosolic protein cloned on the basis of its similarity to Rabphilin3A (28). As the name suggests, Noc2 lacks the C2 domains present in the C-terminal half of Rabphilin3A and yet it is highly homologous (77.9% sequence similarity) to the N-terminal Rab3A-binding domain of Rabphilin. Noc2 appears to be predominantly expressed in endocrine and neuroendocrine tissues, and, although it is also expressed at low levels in brain, it may represent a dense core granule-specific Rab3A effector. Interestingly, despite its homology with Rabphilin3A, an interaction between Noc2 and Rab3A has not been demonstrated (28, 32). Overexpression of Noc2 was found, however, to enhance high K+ induced secretion from transfected PC12 cells, indicating a role for Noc2 in some aspect of regulated secretion from endocrine cell types, although an indirect action on Ca2+ channels or the Ca2+ signal could not be ruled out.

In this study, we have attempted to clarify the function of Noc2 in regulated exocytosis. The data presented here indicate that recombinantly expressed Noc2 is able to specifically associate with Rab3A and that this interaction is sensitive to the guanine nucleotide state of Rab3A. We have also provided evidence for a direct role of Noc2 in regulated exocytosis due to an inhibitory effect of Noc2 overexpression on exocytosis triggered directly by Ca2+ in permeabilized PC12 cells. To determine whether this inhibitory phenotype is dependent directly upon a Noc2-Rab3A interaction, we have generated mutant forms of Noc2, which fail to bind Rab3A completely or which do so with a reduced affinity. Overexpression of the mutant constructs in PC12 cells did not inhibit exocytosis as had been seen with wild type Noc2. We suggest that Noc2 directly associates with Rab3A in neuroendocrine tissues and that this interaction may be essential for an inhibitory action in Ca2+-dependent exocytosis.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of Noc2-- Rat brain cDNA was purchased from Origene (Rockville, MD). The full-length Noc2 sequence was amplified by PCR1 using an Omne-E dryblock thermocycler (Hybaid, Middlesex, United Kingdom (UK)). Primers based on the rat Noc2 nucleotide sequence (GenBankTM accession no. AF022774) containing restriction endonuclease sites (underlined) for subsequent cloning were: sense, 5'-GGCGGGATCCATGGCTGACACCATCTTCAGCAG-3' (BamHI); antisense, 5'-GCCGCTCGAGTCAGTAATGGGTAGTTGCCCAG-3' (XhoI); and antisense, 5'-GCCGAAGCTTTCAGTAATGGGTAGTTGCCCAG-3' (HindIII). For protein expression, BamHI and XhoI primers were used for PCR, the product digested and ligated into pGex4T-2 vector (Amersham Pharmacia Biotech) to generate an N-terminally GST-tagged fusion construct. For expression in PC12 cells, BamHI and HindIII primers were used for PCR and the product ligated into pcDNA3.1(-) (Invitrogen, The Netherlands). All constructs were checked in both directions by automated sequencing (Oswell, Southampton, UK).

Generation of Noc2 Mutants-- Mutations were generated in both pGex4T-2-Noc2 and pcDNA3.1(-)-Noc2 by site-directed mutagenesis using the Quickchange system (Stratagene). For the V58A single amino acid mutant, the primers were: sense, 5'-GGTGGAGGTCATCCTTCAGG(T/C)CATCCAGAGAGCAGAGC-3'; and antisense, 5'-GCTCTGCTCTCTGGATG(A/G)CCTGAAGGATGACCTCCACC-3'. The AAA triple amino acid mutant (residues 154-156) used the following primers: sense, 5'-GGAAGAGGTCAGGGGCCG(T/G)(G/C)G(T/G)(T/C)C(T/G) (A/C)CAAAGGGCTCCCCAAGTAC-3'; and antisense, 5'-GTACTTGGGGAGCCCTTTG(T/G)(A/C)G(A/G)(A/C)C(C/G)(A/C)GGCCCCTGACCTCTTCC-3'.

Nucleotides in parentheses indicate bases that were changed to generate the appropriate amino acid substitutions. The underlined, mutated sequence also generated a NotI restriction site used to screen mutant colonies. All mutant constructs were checked by automated sequencing in both directions (Oswell, Southampton, UK).

Cell Culture and Transfection-- PC12 cells were cultured in suspension in RPMI 1640 media supplemented with 10% (v/v) horse serum, 5% (v/v) fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (37). Cells were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Freshly trypsinized cells were plated onto 24-well trays at a density of 1 × 106 cells/well and transfected with 2 µg of test plasmid along with 3 µg of human growth hormone (hGH) plasmid (pXGH5, Nichols Institute Diagnostics) using LipofectAMINE transfection reagent (Life Technologies, Paisley, UK) as described previously (38, 39). Three days following transfection, the cells were assayed for hGH release. Cells were permeabilized with 20 µM digitonin for 6 min and then challenged with buffer containing either 0 or 10 µM free Ca2+ for 15 min (39). Secreted hGH was quantitated using an enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals, East Sussex, UK) according to manufacturer's instructions.

Transfection of HeLa Cells-- Freshly trypsinized HeLa cells were plated at a density of 100 × 105/35-mm plate and cultured in Dulbecco's modified Eagle's medium + 5% fetal bovine serum. After 24 h, the cells were transfected with 1 µg of plasmid (pcDNA3, or plasmids encoding Noc2, Noc2(V58A), or Noc2(AAA)) using 3 µl of FuGENE transfection reagent (Roche). After an additional 48 h, the cells were lysed in 200 µl of SDS dissociation buffer and samples separated by SDS-PAGE. Nitrocellulose blots were probed with affinity-purified anti-Noc2 (1:100) and visualized using ECL (Amersham Pharmacia Biotech).

Expression and Purification of Recombinant Proteins-- pGex4T-2 Noc2 plasmid constructs encoding wild type, Noc2(V58A), or Noc2(AAA) sequences were used to transform BL-21 RIL codon plus Escherichia coli (Stratagene). Protein expression was induced with 50 µM isopropyl-1-thio-beta -D-galactopyranoside for 1 h. Protein was purified from cytosolic fractions on glutathione-Sepharose 4B (GST-Sepharose, Amersham Pharmacia Biotech) and eluted with 10 mM reduced glutathione. E. coli expressing His6-Rab3A (a gift from Dr. M. Clague, University of Liverpool, UK) were induced with 400 µM isopropyl-1-thio-beta -D-galactopyranoside for 2 h and protein purified from cytosolic fractions on nickel-nitrilotriacetic acid-agarose (Qiagen). Bound protein was eluted using 400 mM imidazole.

GST-Noc2 Binding to Recombinant His6-Rab3A-- 1 µM GST, GST-Noc2, or GST-Noc2 mutant proteins were immobilized onto 20 µl of GST-Sepharose by incubation for 30 min at 4 °C in 100 µl of binding buffer (20 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol, 2 mM MgCl2, 1 mM ATP, 0.5% (v/v) Triton X-100, pH 7.4). This was supplemented with either 50 µM GDP or GTPgamma S. Recombinant His6-Rab3A was then added to incubations at a final concentration of 1 µM and binding allowed to proceed for 2 h at 4 °C with agitation. GST-Sepharose pellets were collected and washed three times in 1 ml of the appropriate binding buffer containing GDP or GTPgamma S. Final pellets were extracted by addition of 50 µl of SDS dissociation buffer (4% (w/v) SDS, 2 mM EDTA, 10% (w/v) sucrose, 1% (v/v) beta -mercaptoethanol, 10% (v/v) glycerol, 125 mM HEPES, pH 6.8) and boiling for 5 min. Bound proteins were analyzed on SDS-PAGE (12.5% gel) and proteins transferred to nitrocellulose membranes for immunoblotting via transverse electrophoresis. Protein was detected with anti-Rab3A (1:400, Transduction Laboratories) and visualized using enhanced chemiluminescence reagents (ECL). In some experiments, the concentration of Rab3A was varied over the range of 0.2-1.0 µM and bound Rab3A quantified using Imagequant densitometry software (Molecular Dynamics).

Analysis of Rat Brain/PC12 Cell Protein Binding to GST-Noc2-- 1 µM GST or GST-Noc2 was immobilized onto 20 µl of GST-Sepharose by incubation for 30 min at 4 °C. 200-µl aliquots of rat brain/PC12 cell cytosolic or membrane proteins, prepared as previously described (40, 41), were then added to incubations and supplemented with either 50 µM GDP or 50 µM GTPgamma S. Binding was allowed to proceed for 2 h at 4 °C with agitation. GST-Sepharose pellets were then collected and washed three times in 1 ml of either GDP- or GTPgamma S-containing binding buffer. Bound proteins were analyzed on SDS-PAGE (12.5% gel) and transferred to nitrocellulose membranes for immunoblotting. Protein was detected using anti-Rab3A (1:400, Transduction Laboratories) or anti-Rab5B (1:100, Santa Cruz Biotechnology) followed by development with ECL.

Production of Noc2 Antiserum-- GST-Noc2 (100 µg) in complete Freund's adjuvant (Pierce, Chester, UK) was injected subcutaneously into rabbits, followed after 4 weeks by a similar injection with incomplete Freund's adjuvant (Pierce). Bleeds were taken thereafter at 2-4-week intervals following boosts with GST-Noc2 (100 µg) in incomplete Freund's adjuvant.

Immunofluorescence-- PC12 cells (1 × 106/well) were plated onto glass coverslips and transiently cotransfected with 2 µg of enhanced green fluorescent protein (GFP) plasmid in combination with 2 µg of pcDNA3.1(-) plasmid (control) or 2 µg of the appropriate pcDNA-Noc2 plasmid construct using LipofectAMINE treatment (Life Technologies, Paisley, UK). Three days following transfection, cells were fixed in 4% formaldehyde in PBS for 30 min, washed twice in PBS, and incubated for 30 min in PBTA (0.1% Triton X-100, 0.3% bovine serum albumin in PBS). Cells were subsequently incubated with anti-Noc2 serum (1:100) in PBTA for 1 h, and washed three times with 1 ml of PBTA. The cells were then incubated with anti-rabbit-biotinylated IgG (1:100, Amersham Pharmacia Biotech) for 1 h and washed three times in 1 ml of PBTA, followed by incubation with streptavidin-Texas Red (1:50, Amersham Pharmacia Biotech) for 30 min. Cells were washed three times with 1 ml of PBTA and coverslips blotted and allowed to air-dry prior to mounting.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Noc2 overexpression has been found to modify K+-induced secretion in PC12 cells but it was not clear if this was a direct effect on exocytosis. We were interested, therefore, to determine whether overexpression of Noc2 would have a direct effect on exocytosis rather than via ion channels, by examining the effect of overexpression on exocytosis in permeabilized cells. The approach used involved cotransfection with a plasmid encoding hGH to allow assay of exocytosis from transfected cells (39, 42). When Noc2 was overexpressed in PC12 cells, we observed a reproducible reduction in hGH release relative to controls (Fig. 1) following stimulation with 10 µM Ca2+ in digitonin-permeabilized cells. It appears, therefore, that Noc2 can exert a direct inhibitory effect on Ca2+-stimulated exocytosis. We also found that Noc2 overexpression led to an inhibition of K+-induced hGH release in intact cells (data not shown).


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Fig. 1.   Overexpression of Noc2 has a direct inhibitory effect upon Ca2+-regulated exocytosis in digitonin-permeabilized PC12 cells. Control vector or plasmid encoding wild-type Noc2 were cotransfected into PC12 cells in combination with a plasmid encoding hGH. Three days following transfection, the cells were permeabilized with digitonin, challenged directly with 0 or 10 µM Ca2+, and released hGH assayed as a percentage of the total hGH expressed. The data shown are combined from three independent transfection experiments with a total n = 18 per condition (mean ± S.E.).

To determine the mechanism by which Noc2 overexpression inhibited secretion we re-analyzed its potential association with Rab3A, as previous studies had apparently failed to show binding between these proteins (28, 32). First, we analyzed the distribution of both Rab3A and the endosome-specific, Rab5B in rat brain protein extracts by immunoblotting, which demonstrated that we could detect Rab3A in the membrane fraction and Rab5B in both membrane and cytosol fractions (Fig. 2A). A series of binding assays were then performed using immobilized GST-Noc2 as an affinity column over which rat brain or PC12 cell protein extracts were passed. In the first of these experiments, rat brain cytosol and membrane protein fractions were incubated with the GST-Noc2 affinity resin, or a GST control column. Incubations were supplemented with either GDP or GTPgamma S, and bound proteins analyzed by immunoblotting. In this experiment (Fig. 2B), GST-Noc2 effectively bound Rab3A from rat brain membrane samples only in the presence of GTPgamma S. As a specificity check, samples were also blotted for the endosomal Rab5B protein. No binding of Rab5B to Noc2 was observed in any sample although Rab5B was easily detectable in the rat brain extracts (Fig. 2A). This result suggests that the Noc2-Rab3A interaction is highly specific since a second closely related Rab protein failed to bind to Noc2 under identical experimental conditions. No detectable binding of Rab3A to GST controls was observed (data not shown). We next used the same affinity binding approach to analyze association of proteins from PC12 cell extracts with immobilized GST-Noc2 (Fig. 2C). In this system, as with rat brain, GST-Noc2 specifically bound Rab3A from membrane protein samples, again in a GTPgamma S-dependent fashion. This interaction was confirmed with purified recombinant Noc2 and Rab3A, where we observed binding that was substantially greater in the presence of GTPgamma S compared with GDP (Fig. 2D). No binding of Rab3A to GST-loaded control beads was detected in the presence of GTPgamma S or GDP (Fig. 2D). This experiment demonstrates that the Rab3A must bind directly to Noc2. To determine the efficiency of binding in the assay, the signal from bound Rab3A was compared with that from known amounts of input Rab3A. From a quantitative analysis, it appeared that around 0.3% of the input was bound. This low efficiency may be due to the low affinity of the Noc2-Rab3A interaction (see below) that would not be well preserved during washing of the beads in the assay.


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Fig. 2.   The guanine nucleotide-dependent interaction of Rab3A with Noc2. A, the distribution of Rab3A and Rab5B in rat brain cytosol and membrane fractions was ascertained by immunoblotting. B, GST-Noc2 was used to affinity-purify protein from rat brain cytosol and membrane extracts in the presence of GDP or GTPgamma S. Immunoblots were then probed with either anti-Rab3A or anti-Rab5B. Duplicates lanes for each condition are presented. GST-Noc2 specifically associated with Rab3A but not Rab5B, from rat brain membranes, in the presence of GTPgamma S. C, GST-Noc2 was used to affinity-purify Rab3A from PC12 extracts in the presence of either GDP or GTPgamma S. Bound Rab3A was visualized using immunoblotting. Noc2 specifically associated with Rab3A from membranes in the presence of GTPgamma S. D, recombinantly expressed GST-Noc2 or GST were incubated with His6-Rab3A in the presence of GDP or GTPgamma S. Rab3A bound to GST-Noc2 was then visualized by immunoblotting. Rab3A binding was much greater in the presence of GTPgamma S. In all of the above binding experiments, no detectable binding of Rab3A or Rab5B to GST in control incubations was observed.

To determine whether the Noc2-mediated inhibition of Ca2+-stimulated exocytosis in PC12 cells depends upon its association with Rab3A, we took a mutagenesis approach, whereby key residues in Noc2 were altered in an attempt to create mutant forms of the protein, which could no longer support binding to Rab3A. Previous studies with Rabphilin3A mutants highlighted a number of residues essential for Rab3A association (31). Based on the high degree of similarity between Noc2 and the Rab3A binding domain of Rabphilin3A (Fig. 3A), we reasoned that mutations at corresponding positions in Noc2 might also generate mutants defective in Rab3A binding. The mutation V61A in Rabphilin3A completely abolishes Rab3A binding and so the first Noc2 mutant generated altered the corresponding valine at position 58 to alanine, to produce Noc2(V58A). The crystal structure of Rab3A complexed to the N-terminal domain of Rabphilin3A has recently been solved (23). From this structural analysis, it is clear that Val-61 of Rabphilin3A (Val-58 of Noc2) contacts the switch region of Rab3A, an important point of interaction between the two proteins. The crystal structure also highlighted the potential importance of a conserved six-amino acid motif, which is found in various Rab3A effector proteins (23). This SGAWF(F/Y) sequence (Fig. 3A) resides in a second important contact area between Rabphilin3A/Rab3A. Mutants in this motif have not previously been examined, but we reasoned from this structural information that mutation of residues in the SGAWF(F/Y) motif should also generate Noc2 mutants with defective Rab3A binding. The second Noc2 mutant generated, Noc2(AAA), therefore changed SGAWFY in Noc2 to SGAAAA. The recombinant Noc2 mutant proteins, fused to GST, were expressed, and their binding to recombinant Rab3A in the presence of GTPgamma S was analyzed and compared with wild type protein (Fig. 3B). Wild type Noc2 effectively associated with Rab3A, as did the Noc2(V58A) single amino acid mutant at high concentrations. Noc2(AAA), conversely, exhibited a complete loss of Rab3A binding in the same assay. Control GST protein gave no detectable Rab3A binding.


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Fig. 3.   Generation of Noc2 mutants and analysis of their binding to recombinant Rab3A. A, alignment of rat Noc2 sequence with the N-terminal Rab3A binding domain of rat Rabphilin3A. The proteins share 40.7% amino acid identity and 77.9% similarity over this sequence. Residues highlighted with boxes define sequences that were chosen for mutation in Noc2 to create Noc2(V58A) and Noc2(AAA). B, recombinant GST-Noc2 protein constructs were incubated with His6-Rab3A in the presence of GTPgamma S and bound Rab was analyzed by immunoblotting. Wild type Noc2 and Noc2(V58A) bound Rab3A; however, the AAA mutation completely abolished the Rab3A association. No Rab3A bound to GST control protein. C, the interaction of Noc2(V58A) was analyzed by an affinity binding assay where a constant (1 µM) concentration of Noc2 was incubated with varying concentrations of Rab3A in the presence of GTPgamma S. Bound Rab3A was visualized by immunoblotting and quantified using densitometry. The data were normalized to maximum binding of wild-type Noc2 protein at 1 µM Rab3A concentration (100% binding). Noc2 (V58A) bound Rab3A but with reduced affinity compared with wild type protein.

To further characterize the binding of the V58A Noc2 mutant, an assay was performed with a constant concentration of Noc2 protein and varying concentrations of wild type or mutant Rab3A in the presence of GTPgamma S (Fig. 3C). In these assays, GST controls, along with GST-Noc2 (AAA) exhibited no detectable binding even at the highest concentration of Rab3A. Wild type Noc2 exhibited saturable binding over a range of Rab3A concentrations. This binding was of lower affinity than observed for Rabphilin3A binding to Rab3A (43) and was half-maximal at around 500 nM Rab3A. Noc2(V58A) also bound Rab3A over this concentration range, although with a lower affinity than the wild type protein and with ~40% less Rab3A bound at the maximal concentration tested (Fig. 3C).

Having generated Noc2 mutants defective in Rab3A association, we next analyzed the effects of overexpression of these proteins on Ca2+-triggered exocytosis from PC12 cells. It was necessary to first ensure that all of the Noc2 constructs were overexpressed in transfected cells, and, for this purpose, an anti-Noc2 antiserum was raised and affinity-purified. On blots with equal loading of recombinant Noc2, Noc2(V58A) and Noc2(AAA), the anti-Noc2 serum recognized each of the proteins with no apparent bias to any particular construct (Fig. 4A). The preimmune antiserum at the same concentrations did not give any signal. To determine that the Noc2 wild-type and the Noc2(V58A) and Noc2(AAA) mutants were expressed at comparable levels and that the full-length protein was expressed in all cases, transfected cells were examined by immunoblotting. The low level of transfection of PC12 cells (around 5%) coupled with the endogenous expression of Noc2 in these cells precludes demonstration of overexpression in this cell type by this technique. We, therefore, adopted a previously used strategy (44) of examining whether the proteins were expressed similarly in an easily transfectable cell type. As shown in Fig. 4B, no endogenous Noc2 was detected in HeLa cells transfected with control vector, and wild-type and mutant Noc2s were present at essentially the same levels, showing that these proteins are expressed and are stable in eukaryotic cells.


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Fig. 4.   Characterization of Noc2 antiserum and confirmation of Noc2 protein overexpression in PC12 cells by immunofluorescence. A, equal loadings of Noc2, Noc2 (V58A), or Noc2(AAA) were immunoblotted with anti-Noc2 serum (1:400). The anti-Noc2 serum recognized each protein with no apparent bias to any particular construct. B, HeLa cells were transfected with control vector (pcDNA3) or plasmids encoding wild-type Noc2, Noc2(V58A), or Noc2(AAA) and blots probed with anit-Noc2. No endogenous Noc2 was detected in control transfected cells, and equivalent amounts of the Noc2 constructs were detected. C, PC12 cells were cotransfected with GFP and each of the Noc2 plasmid constructs. Cells were then stained with anti-Noc2 serum (1:100) and examined using fluorescence microscopy. Control cells transfected with GFP and pcDNA3 expression vector display only background levels of staining with anti-Noc2 (top two panels). Cells transfected with Noc2 or Noc2 mutant expression constructs display elevated levels of anti-Noc2 immunoreactivity in GFP-expressing cells, confirming that all constructs are effectively overexpressed in PC12 cells.

PC12 cells transfected with each of the Noc2 constructs or control vector, in combination with a plasmid encoding enhanced GFP as a marker for transfection, were then processed for immunofluorescence with anti-Noc2. In control conditions with vector, cells expressing GFP displayed only background levels of staining with anti-Noc2 (Fig. 4C, top two panels). Each of the Noc2 expression constructs, by contrast, exhibited anti-Noc2 immunostaining far above background levels in non-GFP-expressing cells (Fig. 4C, bottom six panels). These data confirmed that all Noc2 constructs were efficiently overexpressed in PC12 cells. The effect of overexpression of Noc2(V58A) and Noc2(AAA) on Ca2+-regulated exocytosis was then examined. Overexpression of Noc2(V58A) in PC12 cells had a minor inhibitory effect upon release due to 0 or 10 µM Ca2+ but Ca2+-dependent release was not statistically significantly different (Fig. 5A). Overexpression of Noc2 (AAA) had no distinguishable effect upon Ca2+-dependent hGH release compared with control cells (Fig. 5B). It is apparent that, therefore, these mutants impaired in Rab3A binding are no longer able to support the inhibition of Ca2+-induced secretion elicited by the wild type protein.


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Fig. 5.   The effects of overexpression of Noc2(V58A) and Noc2(AAA) on hGH secretion from PC12 cells. A, PC12 cells were transfected with Noc2(V58A) and hGH release assayed in response to direct Ca2+ challenge after digitonin-permeabilization. Noc2 (V58A) had no significant effect on Ca2+-dependent hGH release compared with control (n = 12 per condition from two transfections, mean ± S.E.). B, overexpression of Noc2(AAA) had no discernible effect on Ca2+-stimulated hGH secretion from digitonin-permeabilized PC12 cells (n = 12 per condition from two transfections, mean ± S.E.).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in this paper have provided the following new insights. First, Noc2 interacts in a nucleotide-dependent manner with Rab3A. Second, Noc2 has a direct regulatory role in Ca2+-dependent exocytosis in permeabilized cells. Third, the demonstration of residues required for Noc2 binding to Rab3A including amino acids within the conserved SGAWFY motif. Fourth, the inhibitory effect of Noc2 overexpression on exocytosis may require interaction with Rab3A as Rab3A binding-deficient mutants were no longer inhibitory.

The importance of Rab GTPases in membrane trafficking processes has long been appreciated (5), although their precise function has remained elusive. Rab3A is unusual in that, unlike Rabs acting at other steps of the secretory pathway (45-47), it appears dispensable for membrane fusion (11, 12). Rab3A knockout mice are viable and display no obvious behavioral abnormalities but display enhanced neurotransmission in response to a nerve impulse (11, 12). An inhibitory role for Rab3A has also been observed in experiments involving endocrine or neuroendocrine cells (8, 17-19). Overexpression of constitutively active, GTPase-deficient, Rab3A mutants inhibits Ca2+-triggered secretion from bovine chromaffin cells (18, 19). In the same cell type, micro-injection of antisense Rab3A oligonucleotides to deplete Rab3A elicited an increased potential to respond to repetitive stimulations, in contrast to wild type cells, which underwent desensitization (18) again consistent with an inhibitory role for Rab3A. In the insulin-secreting HIT-T15 cell line, transfection with GTPase defective Rab3 mutants similarly caused an inhibition of nutrient-triggered insulin release (8, 48).

The regulatory roles of Rab3A in exocytosis are likely to be mediated by one or more specific effector proteins (5). Rab3A effectors appear likely to have a variety of roles, from vesicle tethering and docking with acceptor membranes (14, 29), to cytoskeletal interactions, which perhaps permit vesicles close access to the target membrane, allowing them to subsequently tether and dock (26, 28) as well as inhibitory functions (16). Rab3A interacts with at least two specific effectors, Rabphilin3A and Noc2, which in turn are able to interact with cytoskeletal proteins. Rabphilin3A overexpression enhances secretory responses (30, 49). This effect is not directly mediated by the interaction with Rab3A, as point mutants in Rabphilin3A having defective Rab3A binding still enhance secretion from an endocrine cell line (31). In a reciprocal study (33), Rab3A mutants defective in Rabphilin3A binding retained the wild type ability to inhibit secretion. Further evidence for a Rab3A-independent effect of Rabphilin 3A comes from studies using Rabphilin 3A knockout mice (34). These animals fail to exhibit any of the synaptic aberrations observed in Rab3A knockouts. In addition, Rab3A mutants that fail to bind RIM also retain the ability to inhibit Ca2+-triggered secretion (32). It would therefore seem that there is no link between the negative regulatory effect of Rab3A and these effectors. Rab3A also interacts with Ca2+/calmodulin (35), which has been evoked to explain the inhibitory effect of Rab3A on secretion (32). The Ca2+/calmodulin-Rab3A interaction is, however, difficult to interpret as it displays a number of peculiarities. Ca2+/calmodulin fails to discriminate between GDP and GTP bound forms of Rab3A, arguing against a Rab3A effector function, and its association occurs with much lower affinity than for other specific Rab3A effectors being half-maximal at 20 µM calmodulin (35). In addition, calmodulin has been shown to have positive rather than inhibitory effects on regulated exocytosis (36).

In this study, we have examined the properties of the putative Rab3A effector, Noc2 (28). Noc2 is highly homologous to the Rab3A N-terminal binding domain of Rabphilin3A; however, previous studies have surprisingly failed to detect binding of Noc2 to Rab3A, although no data were shown (28, 32). We have demonstrated a reproducible binding of recombinant Noc2 to Rab3A and furthermore have demonstrated a strict nucleotide dependence of this interaction consistent with an effector function for Noc2 in combination with GTP-bound form of Rab3A. This interaction was also observable when recombinant Rab3A was exchanged for tissue or cell extracts containing a complex mixture of proteins including Rab3A. Noc2 binding was specific for Rab3A, as the endosome-specific Rab5B failed to associate in the presence of GTPgamma S. The affinity of the interaction (~500 nm) was lower than that reported for Rabphilin binding to Rab3A of 150 nM (43) or 20 nM (33). This may explain the low efficiency of binding in the in vitro assay that we observed. The affinity of the interaction of Noc2 with Rab3A was still considerably higher than that reported for Rab3A interaction with calmodulin (35). The reasons for the difference between this and previous studies in detecting Noc2 binding to Rab3A is unclear but may be due to the low affinity of the interaction and to the low stoichiometry of interaction being preserved under in vitro binding conditions.

Noc2 overexpression in PC12 cells resulted in a significant (~50%) inhibition of evoked secretion from permeabilized cells. This differs from previous work showing that overexpression of Noc2 increased K+-induced release from PC12 cells (28). In our hands, K+-induced release of hGH was also inhibited in cells overexpressing Noc2. To test the possibility that this difference was due to a higher level of overexpression in our studies, the plasmid concentration was titrated to search for stimulation at lower plasmid concentrations. The only effect we observed, however, was a reproducible inhibition due to overexpression. The effect of transfection with the Noc2 plasmid appeared to be specific, as we have not seen such a partial inhibition following transfection and use of the GH assay with several other constructs. For example, overexpression of nSec-1 or complexin (38) had no effect on exocytosis, and overexpression of NCS-1 (39) led to an increase in stimulated exocytosis. The only constructs that led to an inhibition in our hands were plasmids encoding clostridial neurotoxin light chains that would be expected to be inhibitory and indeed almost abolished exocytosis (50, 51).

The inhibitory effect of Noc2 overexpression appeared to be dependent upon the Noc2-Rab3A interaction, since mutant forms of Noc2, which lacked the ability to associate with Rab3A, correspondingly lost their ability to inhibit Ca2+-stimulated secretion. The two mutations were in distinct structural regions that interact with Rab3A in Rabphilin 3A (23). We cannot formally rule out, however, that the two mutated domains in Noc2 may also be involved in interactions with some other protein and that the inactivity of the Noc2 mutants could be due to an alternative interaction. One of the tested mutants, Noc2(AAA), also highlights the essential requirement for a conserved Rab effector motif in binding to Rab3A. This motif has been suggested from determination of the crystal structure of the Rab3A/Rabphilin3A complex to be crucial for the interaction. Our mutagenesis of this motif confirms its requirement for Rab3A binding in Noc2. Overall, these data suggest that Noc2 may be a significant Rab3A inhibitory effector in Ca2+-regulated exocytosis of dense-core granules.

    FOOTNOTES

* This work was supported in part by grants from the Wellcome Trust and the Medical Research Council (to R. D. B. and A. M.).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.

Dagger Recipient of a Wellcome Trust Prize studentship award.

§ To whom all correspondence should be addressed. Tel.: 44-151-794-5305; Fax: 44-151-794-5337;E-mail: burgoyne@liverpool.ac.uk.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006959200

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; hGH, human growth hormone; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PBTA, Triton X-100 and bovine serum albumin in phosphate-buffered saline; GTPgamma S, guanosine 5'-O- (thiotriphosphate).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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