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J Biol Chem, Vol. 275, Issue 4, 2938-2942, January 28, 2000

Distinct Protein Domains Are Responsible for the Interaction of Hrs-2 with SNAP-25
THE ROLE OF Hrs-2 IN 7 S COMPLEX FORMATION^*

Susan Tsujimoto and Andrew J. Bean

From the Department of Neurobiology and Anatomy and the W. M. Keck Center for the Neurobiology of Learning and Memory, University of Texas Medical School, Houston, Texas 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulated secretion of neurotransmitter at the synapse is likely to be mediated by dynamic protein interactions involving components of the vesicle (vesicle-associated membrane protein; VAMP) and plasma membrane (syntaxin and synaptosomal associated protein of 25 kDa (SNAP-25)) along with additional molecules that allow for the regulation of this process. Recombinant Hrs-2 interacts with SNAP-25 in a calcium-dependent manner (they dissociate at elevated calcium levels) and inhibits neurotransmitter release. Thus, Hrs-2 has been hypothesized to serve a negative regulatory role in secretion through its interaction with SNAP-25. In this report, we show that Hrs-2 and SNAP-25 interact directly through specific coiled-coil domains in each protein. The presence of syntaxin enhances the binding of Hrs-2 to SNAP-25. Moreover, while both Hrs-2 and VAMP can separately bind to SNAP-25, they cannot bind simultaneously. Additionally, the presence of Hrs-2 reduces the incorporation of VAMP into the syntaxin·SNAP-25·VAMP (7 S) complex. These findings suggest that Hrs-2 may modulate exocytosis by regulating the assembly of a protein complex implicated in membrane fusion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulated release of neurotransmitter at the synapse is necessary for efficient communication between neurons. Calcium transients, produced by membrane depolarization and the subsequent opening of voltage-gated calcium channels, provide the trigger for fusion of the vesicle and plasma membrane lipid bilayers allowing the release of neurotransmitter into the synaptic cleft (1). Biochemical and genetic analyses have identified integral protein components of the vesicle and the plasma membrane along with additional regulatory factors that are hypothesized to be involved in the exocytotic process (2-5).

Synaptosomal associated protein of 25 kDa (SNAP-25)¹ is a component of the core protein complex believed to be central to the exocytotic process (7, 8). SNAP-25 is localized to the plasma membrane, via palmitylation, where it interacts with syntaxin, an integral membrane protein found primarily on the plasma membrane (9-11). Upon binding, syntaxin induces structural changes in SNAP-25 that promote the binding of vesicle-associated membrane protein (VAMP) and the subsequent formation of an SDS-resistant ternary complex that sediments at 7 S (11-14). The ternary complex, recently crystallized, has a structural arrangement consisting of four entwined coiled-coils (15, 16). SNAP-25 contributes two coiled-coils to the complex, while syntaxin and VAMP contribute one coiled-coil each. Although otherwise quite stable when packed together as a four-helix bundle, complex integrity is compromised when SNAP-25 is cleaved with either botulinum neurotoxin type A or E prior to complex formation (17, 18). Such cleavage effectively abolishes neurotransmitter release (13) and suggests that the integrity of the four helical bundle is essential for exocytosis.

We previously identified a novel protein, Hrs-2, that interacts with SNAP-25 (19). The interaction of Hrs-2 with SNAP-25 is inhibited by calcium at concentrations (100 µM) thought to be essential for neurotransmitter release. Moreover, recombinant Hrs-2 inhibits ³H-labeled norepinephrine release from permeabilized PC12 cells (19). Thus, Hrs-2 interacts with a known component of the vesicular trafficking machinery in a calcium-sensitive manner and inhibits calcium-triggered exocytosis, suggesting a role for Hrs-2 in the exocytic machinery.

The 7 S complex (SNAP-25, syntaxin, and VAMP) has been argued to constitute the minimal machinery necessary for membrane fusion (20, 21). However, the molecular mechanisms that regulate complex assembly remain unresolved. Because SNAP-25 is a core constituent of the 7 S complex, we explored the role that Hrs-2 may play in 7 S complex assembly. We identified the sites of interaction between Hrs-2 and SNAP-25 and examined the effects of Hrs-2 on the ability of SNAP-25 to associate with syntaxin or VAMP and the effect of Hrs-2 on 7 S complex assembly.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Restriction enzymes and DNA modifying enzymes were from Promega and Stratagene. Media reagents were from Difco. Materials for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. Horseradish peroxidase-conjugated and ¹²⁵I-labeled anti-rabbit and anti-mouse antisera were obtained from Amersham Pharmacia Biotech. Bovine serum albumin, glutathione-agarose, and Ponceau S were purchased from Sigma. The pGEX-4T1 and pGEX-KG vectors (Amersham Pharmacia Biotech) were used to express recombinant proteins fused to glutathione S-transferase (GST) in the NM522 strain of Escherichia coli. The Hta vector (Life Technologies, Inc.) was used to express recombinant Hrs-2 fused to a His₆ tag via infection of SF21 insect cells.

Plasmid Construction-- Construction of plasmids encoding GST fusion proteins of full-length mouse SNAP-25 (amino acids 1-206), full-length rat syntaxin 1A11 (amino acids 1-288), and full-length rat VAMP2 (amino acids 1-116) were previously described (sn25 in Ref. 19, sytx in Refs. 22 and 23, and VAMP in Ref. 14). A plasmid encoding an carboxyl-terminal truncation of the rat GST-Hrs-2 fusion protein (amino acids 1-478) was prepared by digesting the full-length clone with NheI and BamHI followed by Klenow treatment (30 °C incubation for 10 min) and religation. Two plasmids encoding short and long carboxyl-terminal truncations of the GST-SNAP-25 fusion protein were prepared. The shorter construct, GST-SNAP-25 (amino acids 1-31) was generated by partial restriction digest of the full-length clone with PstI followed by religation. The longer construct, GST-SNAP-25 (amino acids 1-95), was prepared as described (16). Plasmids encoding internal segments of GST-Hrs-2 (coiled-coils 1 and 2 (amino acids 449-562) and coiled-coil 2 (amino acids 478-562)) and the carboxyl-terminal end of GST-SNAP-25 (amino acids 150-206) were prepared by insertion of the appropriate polymerase chain reaction-amplified fragment into pGEX-4T1 (primer sequences available upon request). Constructs generated by restriction digest were sequenced (Sequenase 2.2; U.S. Biochemical Corp.) at the 5'- and 3'-ends to check for proper insertion. All polymerase chain reaction-generated constructs were sequenced in their entirety to ascertain fidelity of sequence.

Expression and Purification of Fusion Proteins-- The His₆-tagged Hrs-2 fusion protein, and all GST fusion proteins were prepared as described (24), with the exception that the recombinant Hrs-2 fusion protein was eluted in batch format using 500 mM imidazole in PBST (phosphate-buffered saline and 0.05% Tween 20). Under these conditions, Hrs-2 is 100% monomeric.² Recombinant SNAP-25, syntaxin 1A11, and VAMP were cleaved from the GST moiety using thrombin (7.5 units/ml; Amersham Pharmacia Biotech) in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl₂, 0.1% -mercaptoethanol. The cleavage reaction was stopped following end-over-end incubation at room temperature for 1 h (syntaxin 1A11), 2 h (VAMP2), or 8 h (SNAP-25) with phenylmethylsulfonyl fluoride (0.1 mM). In some cases, solublized proteins were concentrated using Centricon concentrators (Millipore Corp.). All soluble proteins were precleared with glutathione-agarose prior to quantitation and binding. Protein concentrations were estimated by Coomassie Blue staining of protein bands following SDS-polyacrylamide electrophoresis using bovine serum albumin as a standard.

Western Blotting-- Proteins were resolved on SDS-polyacrylamide gels (12-17% acrylamide) and transferred to nitrocellulose. Blots were stained with Ponceau S in order to ensure accuracy of protein loading, blocked in blotto (5% dry milk in PBST), and incubated with primary antibody diluted in blotto. The following antibodies were used for detection of transferred proteins: 48-5 (Hrs-2 mouse monoclonal antibody, 1:1000 (24), anti-SNAP-25 (mouse monoclonal antibody, 1:1000; obtained from Sternberger Antibodies), HPC-1 (syntaxin mouse monoclonal antibody, 1:1000 (23), and anti-VAMP (rabbit polyclonal, 1:1000, obtained from StressGen Biotechnologies). Filters were washed, and horseradish peroxidase- or ¹²⁵I-conjugated secondary antibody was applied. Antibodies were visualized using either enhanced chemiluminescence (Pierce) or PhosphorImager analysis (Molecular Dynamics).

In Vitro Binding Assays-- For domain mapping experiments, 2 µg of SNAP-25 (Fig. 1) or Hrs-2 (Figs. 2 and 3) and 0.5 µg of various GST fusion proteins immobilized on glutathione-agarose were incubated in binding buffer (20 mM HEPES, pH 7.4, 150 mM KCl, and 0.05% Tween 20) to a final reaction volume of 35 µl. Following an end-over-end incubation at 4 °C for 1 h, samples were washed three times with 150 µl of binding buffer, solublized in SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to immunoblot analysis using enhanced chemiluminescence.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Identification of the domain of Hrs-2 necessary for binding to SNAP-25. Three GST-Hrs-2 fusion proteins encompassing residues 1-478, 449-562, and 478-563 (depicted in the schematic diagram, top) were generated by bacterial expression, immobilized on glutathione-agarose beads, and assayed for binding interactions with full-length SNAP-25. SNAP-25 binds to Hrs-2 truncation mutants that contain the second of two coiled-coil regions (lanes 3 and 4). Neither GST control beads (lane 1) nor the Hrs-2 truncation mutant containing a FYVE-type zinc finger and the first of the two coiled-coil regions (lane 2) were able to bind SNAP-25. These data suggest that the second coiled-coil of Hrs-2 is necessary and sufficient to bind to SNAP-25.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Identification of the domain of SNAP-25 necessary for binding to Hrs-2. Three GST-SNAP-25 fusion proteins, encompassing residues 1-95, 1-31, and 150-206, in addition to full-length GST-SNAP-25 (depicted in the schematic diagram, top) were generated by bacterial expression, immobilized on glutathione-agarose beads, and assayed for binding interactions with full-length Hrs-2. Hrs-2 binds to both full-length SNAP-25 and a SNAP-25 truncation mutant that contains the first two coiled-coil regions (lanes 2 and 3). Neither GST control beads (lane 1) nor the SNAP-25 truncation mutants containing the first or the third coiled-coil regions (lanes 4 and 5) were able to bind Hrs-2. Thus, the second coiled-coil of SNAP-25 (residues 32-95) is required for Hrs-2 binding.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Binding interaction of Hrs-2 with members of the 7 S core complex. Glutathione S-transferase fusion proteins containing glutathione S-transferase (control, lane 1), full-length SNAP-25 (lane 2), VAMP2 (lane 3), and syntaxin 1A11 (lane 4) were prepared as described under "Experimental Procedures" and immobilized on glutathione-agarose beads. Hrs-2 physically interacts with SNAP-25 but not with either syntaxin 1A11 or VAMP2.

To understand the effects of Hrs-2 on complex formation (Figs. 4 and 5), Hrs-2 (0-10 µM) or syntaxin 1A11 (0-3.2 µM) and 0.2 µM GST-SNAP-25 immobilized on glutathione-agarose were mixed in binding buffer and incubated as described above. VAMP2 was added to the GST-SNAP-25/Hrs-2 reactions in a final concentration of 4 µM, and 2 µM Hrs-2 was added to the GST-SNAP-25/syntaxin 1A11 reactions. As a control, a second set of GST-SNAP-25/syntaxin 1A11 reactions to which no Hrs-2 was added was processed simultaneously. The reactions were incubated end-over-end, at 4° C for an additional 1 h and then processed as described above with the exception that ¹²⁵I-conjugated secondary antibody and PhosphorImager analysis were used to analyze the immunoblots. GST-SNAP-25 (bottom panels of Figs. 4 and 5) was stained with Ponceau S to determine the accuracy of loading. Quantitation of antibody-detected protein binding is reported in optical density units.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Syntaxin 1A11 potentiates Hrs-2 binding to SNAP-25. A, quantitation of syntaxin 1A11 () bound to SNAP-25 in the presence (left) and absence (right) of Hrs-2 () (expressed as optical density) versus concentration of syntaxin added in the reaction. B, a constant amount of immobilized GST-SNAP-25 (0.2 µM, lower panels) was incubated with varying amounts of syntaxin 1A11 (0-3.2 µM, middle panels) in the presence (upper left panel) or absence (upper right panel) of a constant amount of Hrs-2 (2 µM). Samples were processed, and immunoblots were visualized as described under "Experimental Procedures." For Hrs-2 binding in the presence of GST-SNAP-25/syntaxin 1A11, the pixel values (× 1000) for the five concentrations of syntaxin 1A11 were 0, 0.8, 1.6, 4.1, and 5.2 (left panel) and 1.0, 0.2, 0.8, 3.5, and 3.4 (right panel). The corresponding pixel values (× 1000) for Hrs-2 were 50.2, 94.0, 111.9, 106.7, and 107.3 (left panel) and 0, 0, 0, 0, and 0 (right panel).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Hrs-2 interferes with VAMP2 binding to SNAP-25. A, the quantitation of VAMP2 () and Hrs-2 () bound to SNAP-25 (expressed in optical density units) versus concentration of syntaxin in the reaction. B, a constant amount of immobilized GST-SNAP-25 (0.2 µM, lower panel) was incubated with varying amounts of Hrs-2 (0-10 µM, middle panel) and a constant amount of VAMP2 (4 µM, upper panel). Samples were processed, and immunoblots were visualized as described under "Experimental Procedures." For VAMP binding in the presence of GST-SNAP-25·Hrs-2 (Fig. 5), the pixel values (× 1000) for the six concentrations of Hrs-2 were 0, 1.4, 7.4, 82.9, 108.6, and 102.3. The corresponding pixel values (× 1000) for VAMP were 5.8, 4.9, 5.0, 0.7, 2.0, and 0.9.

To examine the effect of Hrs-2 on the efficiency of 7 S complex formation (Fig. 6), GST-syntaxin (1.5 µM, glutathione-agarose-immobilized), SNAP-25 (1.5 µM, soluble), and excess VAMP (5.5 µM, soluble) were incubated in the presence of increasing amounts of soluble Hrs-2 (0, 1.5, 3.0, 4.5, and 6.0 µM). The reactants were mixed together in binding buffer (25-µl final reaction volume), incubated end-over-end at 4 °C for 1.5 h, and then processed for quantitation by PhosphorImager analysis as described above. Initial attempts at forming the 7 S complex on glutathione-immobilized SNAP-25 were unsuccessful; therefore, glutathione-immobilized syntaxin was used in all subsequent reactions requiring 7 S complex formation. Ponceau S stain of the GST-syntaxin (bottom panel) was used to determine accuracy of protein loading. Quantitation of antibody-detected protein binding is reported in optical density units.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Hrs-2 decreases the efficiency of 7 S complex formation. Quantitation of the amount of Hrs-2 (A) or VAMP (B) incorporated into the 7 S complex (expressed in optical density units) in the presence of increasing amounts (0, 1.5, 3.0, 4.5, and 6.0 µM) of Hrs-2 is shown. C, glutathione-immobilized GST-syntaxin, SNAP-25, and VAMP were incubated in the presence of increasing amounts of Hrs-2. Samples were processed, and immunoblots were visualized as described under "Experimental Procedures." For the five concentrations of Hrs-2, the pixel values (×1000) for VAMP incorporated into the 7 S complex were 5.5, 2.3, 3.3, 2.2, and 1.4. The corresponding pixel values (× 1000) for Hrs-2 bound were 0, 25.6, 58.1, 55.4, and 56.8.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To determine the domain of Hrs-2 that binds to SNAP-25, we constructed a set of GST fusion proteins containing regions of Hrs-2 predicted to form structural motifs (Fig. 1, top). These truncation mutants were expressed, bound to glutathione-agarose beads, and then examined for interaction with full-length soluble SNAP-25 during an in vitro binding assay. As shown in Fig. 1 (bottom), SNAP-25 bound to Hrs-2 truncations that contained the second of two coiled-coil regions and not to GST beads alone or to an Hrs-2 truncation mutant missing the second coiled-coil region. Since SNAP-25 was able to bind to the Hrs-2 truncation containing only the second coiled-coil, we conclude that the second coiled-coil of Hrs-2 is both necessary and sufficient for binding to SNAP-25.

In a similar manner, we determined the domain of SNAP-25 that binds to Hrs-2. GST fusion proteins containing coiled-coil regions of SNAP-25 were constructed, expressed, immobilized on glutathione-agarose, and then assayed for interaction with full-length soluble Hrs-2. Both full-length SNAP-25 and an amino-terminal truncation construct containing the first two coiled-coils were able to bind Hrs-2. Neither a construct containing the first nor one containing the third coiled-coil was able to interact with Hrs-2. Thus, we conclude that the SNAP-25 binding domain necessary for interaction with Hrs-2 contains residues 32-95, the region encompassing the second coiled-coil motif.

Since SNAP-25 has been previously shown to interact with both syntaxin 1A and VAMP, we examined whether Hrs-2, when in complex with SNAP-25, might affect the ability of SNAP-25 to interact with either syntaxin or VAMP. Hrs-2 did not physically interact with either syntaxin or VAMP (Fig. 3). Therefore, any effect Hrs-2 might have on the ability of SNAP-25 to interact with syntaxin 1A or VAMP would be consequent to the interaction of Hrs-2 with SNAP-25.

We first examined the ability of syntaxin 1A to associate with GST-SNAP-25 when Hrs-2 was present. As a control, we examined the association of GST-SNAP-25 with Hrs-2 in the absence of syntaxin 1A. At all concentrations, Hrs-2 did not affect the amount of syntaxin able to bind to SNAP-25 (Fig. 4). However, we observed that the amount of Hrs-2 bound to SNAP-25 in the presence of syntaxin was greater than the amount bound in its absence, suggesting that syntaxin facilitates the binding of Hrs-2 to SNAP-25 (data not shown). To understand whether this facilitation was dependent on syntaxin, a constant concentration of Hrs-2 was incubated with GST-SNAP-25 in the presence of increasing concentrations of syntaxin (Fig. 4). As the concentration of syntaxin was increased, we observed more Hrs-2 binding. These data demonstrate that syntaxin binding to SNAP-25 enhances the ability of Hrs-2 to associate with SNAP-25.

The effect of Hrs-2 on VAMP binding to SNAP-25 was examined by incubating increasing concentrations of Hrs-2 in a reaction containing VAMP and GST-SNAP-25 (Fig. 5). As the concentration of Hrs-2 was increased, the ability of VAMP to bind to GST-SNAP-25 was diminished in a concentration-dependent manner. Since Hrs-2 has a lower EC₅₀ for SNAP-25 than VAMP, even in the presence of syntaxin (Table I), these data suggest that Hrs-2 may interfere with VAMP assembly into the 7 S core complex.

We next examined the effect of Hrs-2 on 7 S complex assembly (Fig. 6). The trimeric reaction involving Hrs-2, SNAP-25, and VAMP demonstrated that Hrs-2 interferes with the ability of VAMP to bind to SNAP-25. The quaternary reaction involving Hrs-2 and the three 7 S complex binding partners (GST-SNAP-25, syntaxin, and VAMP) reveals that when Hrs-2 is added to the 7 S complex in increasing concentrations, the amount of VAMP incorporated into the 7 S complex decreases. When the concentration of Hrs-2 reaches binding saturation, VAMP incorporation is reduced by 80%.

It is possible that the 7 S complex still forms despite the presence of Hrs-2, albeit with greatly reduced efficiency. Alternatively, the 7 S complex may not form at all, and the small amount of VAMP we detect is only associated with syntaxin. Additional studies will be needed to clarify the precise structural impact of Hrs-2 on the 7 S complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent evidence suggests a role for Hrs-2 in the secretory machinery. Hrs-2 physically interacts in a Ca²⁺-dependent manner with SNAP-25, a component of a core protein complex thought to be essential for neurotransmitter release. (19). Additionally, both Hrs-2 and SNAP-25 are present in nerve terminals as demonstrated by light (24) and electron microscopic studies³ as well as biochemical fractionation studies.⁴ Moreover, recombinant Hrs-2 inhibits secretion in permeabilized PC-12 cells (19). In the present study, we have identified the protein domains mediating the interaction between SNAP-25 and Hrs-2. We show that Hrs-2 and SNAP-25 interact through regions that are predicted to form -helical coiled-coils on both proteins. We have also examined the effect of Hrs-2 on other SNAP-25-interacting proteins. The interaction of Hrs-2 with SNAP-25 is enhanced in the presence of syntaxin. Furthermore, either Hrs-2 or VAMP may bind to SNAP-25; however, they cannot bind simultaneously. Additionally, increasing amounts of Hrs-2 added to the 7S complex results in a disassociation of VAMP from that complex. These data suggest that Hrs-2 may be involved in regulating the formation of the 7 S protein complex through interactions with SNAP-25.

-Helical coiled-coils are a common structural motif through which protein-protein interactions can occur (26, 27). Hrs-2 contains two regions predicted to form coiled-coils (19), and we show that SNAP-25 binds to the second coiled-coil of Hrs-2 (Fig. 1). SNAP-25 is predicted to form three coiled-coils (10). An ordered coiled-coil structure over the first two coiled-coils of SNAP-25 is induced when syntaxin interacts with the amino terminus of SNAP-25 (11, 12). In addition, the carboxyl-terminal region of SNAP-25 also becomes structured, most likely forming a third coiled-coil (11, 13). Like syntaxin, Hrs-2 may also transform the structure of SNAP-25 such that the ability of SNAP-25 to associate with other proteins is altered.

Within the context of the 7 S complex, VAMP interacts with the third coiled-coil of SNAP-25. Thus, although VAMP and Hrs-2 are not competing for the same binding site on SNAP-25, the presence of Hrs-2 inhibits VAMP binding. The inhibition of VAMP binding may result from Hrs-2 binding to SNAP-25. The interaction of Hrs-2 with SNAP-25 may fail to promote an ordered structure in the third coiled-coil region of SNAP-25 where VAMP binds (12). An alternative possibility is that steric hindrance due to the binding of Hrs-2 prevents VAMP binding.

Our results show that formation of the SNAP-25·syntaxin complex enhances the binding of Hrs-2 to SNAP-25. The binding of Hrs-2 to the syntaxin·SNAP-25 complex may be favored over VAMP binding, since Hrs-2 has a higher binding EC₅₀ for this complex. The presence of Hrs-2 effectively blocks VAMP from interacting with SNAP-25. Moreover, Hrs-2 appears to prevent the formation of the 7 S complex by competing with VAMP for entry into the complex. It is possible that the 7 S complex still forms despite the presence of Hrs-2, albeit with greatly reduced efficiency. Alternatively, under these conditions the 7 S complex may not form at all, although a small amount of VAMP remains able to associate with the syntaxin that is not present in the 7 S complex. Additional studies will be needed to clarify the precise structural impact of Hrs-2 on the 7 S complex.

The present data indicate that Hrs-2 has a regulatory role on the exocytic machinery and suggest a heuristic model whereby following an influx of calcium, a conformational change in Hrs-2 may dissociate Hrs-2 from SNAP-25, allowing VAMP to bind and the 7 S complex to assemble. This model predicts that Hrs-2 would inhibit secretion when present prior to formation of the 7 S complex. In permeabilized PC12 cells, Hrs-2 inhibits secretion of ³H-labeled norepinephrine when added during the Mg-ATP-dependent "priming" step (19). This step precedes Ca²⁺-dependent fusion and has been shown to correlate with an ATP-dependent catalysis of the 20 S complex that leaves only the SNAP-25/syntaxin interaction intact (28). The addition of exogenous Hrs-2 at this stage in the assay may compete for VAMP binding and inhibit ³H-labeled norepinephrine release. Thus, Hrs-2 appears to act prior to the "priming" step in the vesicle life cycle, which may correspond to the movement of vesicles from an endocytic/sorting/recycling phase to a releasable state.

	ACKNOWLEDGEMENTS

We thank Yuchieh Chang and Bill Evans for contributing to these studies. We also thank Drs. Shu-Chan Hsu, Karen Ervin, Neal Waxham, and Tom Vida for helpful discussions and Drs. Waxham and Vida for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by the Mallinckrodt Foundation and National Institutes of Health Grant MH058920.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.

To whom correspondence should be addressed: Dept. of Neurobiology and Anatomy, University of Texas Medical School, 6431 Fannin St., Rm. 7.208, Houston, TX 77030. Tel.: 713-500-5614; Fax: 713-500-0623; E-mail: abean@nba19.med.uth.tmc.edu.

² S. Tsujimoto and A.J. Bean, unpublished observations.

³ S. Davanger and A.J. Bean, unpublished observations.

⁴ A. J. Bean, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: SNAP-25, synaptosomal associated protein of 25 kDa; VAMP, vesicle-associated membrane protein; Hrs-2, hepatocyte growth factor-regulated tyrosine kinase substrate 2; GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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