WO1998038210A2 - Methods and compositions for modulation of vesicular release - Google Patents

Abstract

Hrs-2 polynucleotides and polypeptides are disclosed. Also disclosed are methods of identifying a compound capable of modulating calcium-regulated secretion of secretory vesicles, such as the release of neurotransmitter-containing synaptic vesicles.

Description

Methods and Compositions for Modulation of Vesicular Release

Field of the Invention

The present invention relates to methods and compositions useful for identifying compounds capable of modulating vesicular release. In particular, the invention relates to (i) the Hrs-2 ATPase, and (ii) methods of identifying compounds capable of modulating vesicular release employing the Hrs- 2 ATPase.

References

Ausubel, F.M., et al. in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media, PA (1988).

Bartel, P., et al. , BioTechniques 14:920-924 (1993). Bennett, M.K., et al.. Cell 74:863 (1993).

Bennett, M.K. and Scheller, R.H., Ann. Rev. Biochemistry 63:63-100 (1994).

Bourne, H.R., et al. , Nature 349: 117-127 (1991).

Brent, R., et al. , Cell, 43:729-736 (1985).

Chapman, E.R., et a/. , J. Biol. Chem. 269:27427-27432 (1994). Chenchik, A., et al., Clontechniques X(l):5-8 (1995).

Cheng, Y.-S.E., et al., Mol. Cell. Biol. J .:4717-4725 (1991).

Chien, C.-t, et al. , Proc. Natl. Acad. Sci. U.S.A. , 88:9578 (1991).

Dever, T.E., et al. , Proc. Natl. Acad. Sci. USA 84: 1814-1818 (1987).

Durfee, T., et al. , Genes & Development 2:555 (1993). Elferink, L.A., et al. , Cell 72: 153 (1993).

Fields, S., and Song, O., Nature 340:245-246 (1989).

Gietz, R.D., and Schiestl, R.H. , Yeast 7:253-263 (1991).

Guan, K.L. and Dixon, J.E., Anal. Biochem. 192:262 (1991).

Gyuris J., et al. , Cell, 75:791-803 (1993). Harlow, E., et al., ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor Laboratory

Press (1988).

Hay, J.C., and Martin, T.F., J. Cell. Biol. .119: 139-151 (1992).

Kemler, R., Trends Genet. 9:317-321 (1993).

Komada, M., and Kitamura, N., Mol. Cell. Biol. 15:6213-21 (1995). Kozak, M., Cell 44:283-292 (1986). Lomneth, R., et al., J. Neurochem. 57:1413-1421 (1991).

Malgaroli, A., and Tsien, R.W., Nature 357:134-139 (1992).

Morgan, A., et al. , J. Biol. Chem. 269:29347-29350 (1994).

Mullis, K.B., et al., U.S. Patent No. 4,683,195, issued 28 July 1987. Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987.

Oyler, G.A., et al. J. Cell. Biol. 109:3039-3052 (1989).

Parsons, T.D., et al.. Neuron 15:1085-1096 (1995).

Pevsner, J., et al. Neuron 13:353-361 (1994).

Rothman, J.E., Nature 372:55-63 (1994). Sambrook, ., etal.. MOLECULAR CLONING: A LABORATORY MANUAL. Second Edition, Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

Scheller, R.H., Neuron 14:893-897 (1995).

Smith, S., and Augustine. G., ϋ:458-464 (1988).

Smith, D.B., and Johnson, K.S., Gene 67:31 (1988). Sόllner, T., et al., Nature 362:318-324 (1993a).

Sollner, T., et al., Cell 75:409-418 (1993b).

Sϋdhof, T.C., Nature 375:645-653 (1995).

Tagaya, M., et al. , J. Biol. Chem. 268:2662-2666 (1993).

Ting, A.E., et al., Proc. Natl. Acad. Sci. USA 92:9613-9617 (1995). Whiteheart, S.W., et al., J. Cell. Biol. 126:945-954 (1994).

Background of the Invention

Signal transmission between nerve cells typically involves the release of neurotransmitter from a presynaptic cell onto a postsynaptic cell. The neurotransmitter in the presynaptic cell is contained in synaptic vesicles positioned above the release sites at the presynaptic membrane (active zones). In response to a release signal (typically a local influx of calcium due to a depolarization of the presynaptic terminal), the vesicles undergo a series of mobilization steps culminating in the fusion of the vesicles with the presynaptic terminal membrane, and a dumping of vesicle contents into the synaptic cleft. The neurotransmitter molecules diffuse across the synaptic cleft and bind to corresponding receptors in the postsynaptic membrane to communicate the appropriate signal (typically a depolarization or hyperpolarization of the postsynaptic membrane) to the postsynaptic cell. Much of the neurotransmitter in the synapse is subsequently re-absorbed by the presynaptic cell through specific transmitter uptake mechanisms. A number of drugs affecting signalling in the central nervous system (CNS) and the peripheral nervous system (PNS) have been developed. Some of these, such as phenoxybenzamine, block specific post-synaptic receptors; others, such as clonidine and diethylamide, stimulate such receptors; still others (e.g., desipramine, imipramine) act on reuptake mechanisms, and some act on neurotransmitter synthesis (e.g., α-Methyltyrosine, p-Chlorophenylalanine) or degradation (e.g., monoamine oxidase inhibitors, iproniazid, pargyline).

The present invention provides a tool for the screening and identification of drugs capable of affecting secretory processes, such as neurotransmitter release at the active zones of presynaptic membranes.

Summary of the Invention

In one aspect, the present invention includes a substantially purified Hrs-2 polypeptide, such as a polypeptide encoded by a polynucleotide sequence derived from the genome of a mammal (e.g. , a rat or a human). In one embodiment, the polypeptide contains a region of at least 6, preferably at least 8, more preferably at least 10 or more consecutive amino acids corresponding to a region contained in SEQ ID NO:4 or SEQ ID NO:6. An exemplary rat Hrs-2 polypeptide contains the sequence represented as SEQ ID NO:4. An exemplary human Hrs-2 polypeptide contains the sequence represented as SEQ ID NO:6. In another aspect, the invention includes a substantially purified Hrs-2 polynucleotide, such as a polynucleotide having a sequence derived from the genome of a mammal (e.g. , a rat or a human).

In one embodiment, the polynucleotide contains a region of at least 12, preferably at least 16, more preferably at least 20 or more consecutive nucleotides corresponding to a region contained in

SEQ ID NO: 3 or SEQ ID NO:5. An exemplary rat Hrs-2 polynucleotide contains the sequence represented as SEQ ID NO: 3. An exemplary human Hrs-2 polynucleotide contains the sequence represented as SEQ ID NO:5.

The invention also includes a method of identifying a compound capable of modulating (e.g. , potentiating or inhibiting) calcium-regulated secretion of secretory vesicles (e.g. , release of neurotransmitter-containing synaptic vesicles). The method includes the steps of: (i) contacting a SNAP-25 polypeptide (e.g. , a SNAP-25 polypeptide having the sequence of SEQ ID NO: 12) with an Hrs-2 polypeptide (e.g. , an Hrs-2 polypeptide having a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6), in the presence and absence of a test compound, (ii) measuring the effect of the test compound on the extent of binding between the SNAP-25 and Hrs-2 polypeptides, and (iii) identifying the compound as effective if its measured effect on the extent of binding is above a threshold level. In one general embodiment, the test compound is capable of inhibiting the binding between the SNAP-25 and Hrs-2 polypeptides. In another general embodiment, the test compound is capable of potentiating the binding between the SNAP-25 and Hrs-2 polypeptides. The contacting may include contacting an Hrs-2 that is immobilized on a solid support, or it may include contacting a SNAP-25 polypeptide that is immobilized on a solid support.

The method may be used, for example, to assay small molecule test compounds contained in a small molecule combinatorial library, peptide test compounds (e.g. , from a combinatorial peptide library), a library of toxins, such as conotoxins, and the like.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.

Brief Description of the Figures

Fig. 1 is a schematic illustration of the domain structure of Hrs-2.

Fig. 2 A shows Hrs-2 binding to GST-SNAP-25 immobilized on glutathione-agarose beads and inhibition of the binding by Zn²⁺ . Fig. 2A is plot of the binding as a function of Hrs-2 concentration.

Figs. 2B shows binding of Hrs-2 to immobilized GST-SNAP-25 in the absence and presence of increasing concentrations of free calcium. Fig. 2B is plot of the binding as a function of free calcium concentration.

Fig. 3A is the raw data and plot showing that recombinant Hrs-2 elutes from a gel filtration column in two peaks.

Fig. 3B is a plot showing the NTPase activity of monomeric Hrs-2.

Fig. 3C is a plot showing the NTPase activity of oligomeric Hrs-2.

Fig. 3D is a plot showing the inhibition of the ATPase activity of monomeric Hrs-2 by ATPγS. Fig. 3E is a plot showing the inhibition of the ATPase activity of monomeric Hrs-2 by

GTPγS.

Figs. 4A-D are computer-generated image of immunohistochemical localization of Hrs-2 in cultured hippocampal neurons.

Fig. 5 is a plot showing dose-dependent and saturable inhibition of the release of ³H- norepinephrine (NE) from permeabilized PC 12 cells by Hrs-2.

Figures 6 A and 6B show fragmentary plan views of control (Fig. 6 A) and experimental (Fig. 6B) multiwell plates used in a biochemical binding assay to identify compounds affecting binding of Hrs-2 to SNAP-25. Detailed Description of the Invention

I. Definitions

"Substantially purified" refers to the at least partial purification of a selected polynucleotide, polypeptide, antibody or related compound away from unrelated or contaminating components (e.g. , serum cells, other proteins).

When a first polynucleotide fragment or polypeptide fragment is said to "correspond to" a second polynucleotide fragment or polypeptide fragment, respectively, it means that the fragments or regions are essentially co-extensive with one another when the sequences representing the fragments are aligned using a sequence alignment program, such as "MACVECTOR" (IBI, New Haven, CT). "Corresponding" polynucleotide or polypeptide fragments typically contain a similar, if not identical, number of residues. It will be understood, however, that corresponding fragments may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences.

The term "significant" , when used with reference to "significantly different", "significantly inhibits" or "significantly stimulates", refers to a difference in a quantifiable parameter between the two groups being compared that is statistically-significant using standard statistical tests. For example, the degree of binding in a protein binding assay may be quantified using standard methods, and the degree of binding under different conditions can be compared for statistically-significant differences.

An antibody or antibody composition (e.g. , polyclonal antibodies) is "specifically immunoreactive" with a selected protein when the antibody or antibody composition is not reactive with antigens typically present in normal sera, not exposed to the selected protein.

A polypeptide is "characterized by" a selected sequence when the polypeptide contains a sequence that is identical or substantially identical to the selected sequence.

"SNAP-25" refers to synaptosomal-associated protein of 25 kDa (Pevsner, et al., 1994; Scheller, 1995).

"α-SNAP" refers to soluble NSF attachment protein (Pevsner, et al., 1994; Scheller, 1995). "NSF" refers to N-ethylmaleimide-sensitive factor (Pevsner, et al., 1994; Scheller, 1995). "VAMP" refers to vesicle-associated membrane protein (Pevsner, et al., 1994; Scheller, 1995). "VAMP" refers to vesicle-associated membrane protein (Pevsner, et al., 1994; Scheller,

1995). II. Overview of the Invention

The present invention relates to methods and compositions effective to modulate neurotransmitter release. The invention is based on the discovery, described herein, of Hrs-2 - a novel ATP-preferring nucleotidase that associates with SNAP-25, a component of the protein polypeptides thought to underlie vesicle docking and fusion. Experiments performed in support of the invention demonstrate, among other things, that (i) the binding of recombinant Hrs-2 protein to SNAP- 25 is reduced by addition of calcium in the concentration range that supports neurotransmission (Smith and Augustine, 1988), and (ii) Hrs-2 dose-dependently inhibits calcium-triggered ³H-NE release from permeabilized PC12 cells. The results support a role for Hrs-2 in regulated secretion via calcium regulated interactions with SNAP-25 (Hay and Martin, 1992; Parsons, et al. , 1995).

III. Secretion

All eucaryotic cells possess transport vesicles, which continuously carry new plasma membrane components to the plasma membrane from the Golgi apparatus and incorporate such components into the plasma membrane through a process termed vesicle fusion. Eucaryotic cells also typically secrete various types of molecules through a process termed exocytosis, whereby the contents of the transport vesicles are released outside of the cell during the vesicle fusion process. There are two basic types of exocytosis — constitutive and regulated.

Constitutive exocytosis refers to the process whereby the proteins and/or other components destined for secretion are packaged in the Golgi apparatus into transport vesicles, which are then promptly transported to and fused with the plasma membrane.

Regulated exocytosis refers to the type of exocytosis where the vesicles are stored in the cell and released only when triggered by some extrinsic event, such as the influx of calcium (as happens, e.g., at a neural synapse in the case of neurotransmitter release), or the binding of a ligand to a receptor (as happens, e.g., in the case of histamine release from mast cells).

An exemplary system in which to study regulated exocytosis is chemical synaptic transmission in neurons. Neurons communicate with target cells through regulated exocytosis of chemical messengers (reviews: Scheller, 1995; Sϋdhof, 1995). Within the presynaptic terminal, neurotransmitter is packed into synaptic vesicles which are targeted to active zones at the nerve terminal plasma membrane. Action potential-induced elevation of intracellular calcium increases the probability of fusion between synaptic vesicles and the plasma membrane lipid bilayer, resulting in the release of neurotransmitter into the synaptic cleft.

Associations between proteins present on neurotransmitter-containing vesicles and on the presynaptic membrane are proposed to underlie docking and fusion of synaptic vesicles with the plasma membrane, obligate steps in regulated neurotransmission (Bennett and Scheller, 1994; Scheller, 1995; Sollner, et al. , 1993a, 1993b). Such synaptic transmission proceeds through a series of vesicle mobilization steps involving specific proteins. The targeting of a vesicle to the appropriate acceptor membrane occurs through the formation of a 7S polypeptide, which is comprised of the two vesicle proteins, VAMP and synaptotagmin, along with the two target membrane proteins, SNAP-25 and syntaxin (Bennett and Scheller, 1994; Scheller, 1995; Sollner, et al. , 1993a, 1993b; Chapman, et al. , 1994; Sudhof, 1995; Pevsner, et al , 1994; Rothman, 1994; Oyler, et al , 1989). The vesicle fusion process is then thought to progress with the addition of αSNAP to the 7S polypeptide, followed by the binding of NSF to form the 20S particle, consisting of syntaxin, VAMP, SNAP-25, α-SNAP and NSF. To form a stable 20S polypeptide, non- hydrolyzable forms of ATP must be bound to NSF. Upon ATP hydrolysis by NSF, the 20S polypeptide dissociates into its component subunits and the vesicle fuses with the target membrane.

Experiments performed in support of the present invention and described in detail below have resulted in the identification of additional proteins that are involved in both regulated and constitutive exocytosis. Specifically, a novel protein (termed Hrs-2) that interacts with SNAP-25 has been discovered. The binding of Hrs-2 to SNAP-25 is inhibited by calcium in the physiological concentration range that supports synaptic transmission. Furthermore, Hrs-2 binds and hydrolyzes nucleoside triphosphates with kinetics suggesting that ATP is the physiological substrate for this enzyme. Hrs-2 is expressed broadly throughout the brain, is present in nerve terminals, and recombinant Hrs-2 inhibits calcium-triggered ³H-norepinephrine release from permeabilized PC 12 cells. These data suggest a role for Hrs-2 in regulating secretory processes through calcium- and nucleotide-dependent modulation of vesicle-trafficking protein polypeptides.

IV. Hrs-2 Polynucleotides and Polypeptides

Experiments performed in support of the present invention have resulted in the identification and isolation of polynucleotide sequences encoding a novel protein termed Hrs-2. The invention includes, in one aspect, such substantially isolated or substantially purified Hrs-2 polynucleotide sequences. Such Hrs-2 sequences can be isolated, for example, from cDNA mammalian cDNA libraries, as described in Example 1. Exemplary Hrs-2 sequences include the rat sequence (SEQ ID NO:3 and a partial human sequence (SEQ ID NO:5). The human sequence is missing the portion encoding the amino terminal 149 amino acids of the protein.

Full-length sequence information can be obtained using standard methods (e.g., Sambrook, et al. , 1989; Ausubel, et al. , 1988). For example, the sequences provided herein may be used to design forward and reverse polymerase chain reaction (PCR) primers, which can be employed to amplify longer cDNA fragments (Mullis, 1987; Mullis, et al. , 1987) for library screening or "rapid amplification of cDNA ends" (RACE) PCR (Chenchik, et al., 1995). DNA or cDNA libraries may be made according to established methods (Ausubel, et al. , 1988; Sambrook, et al. , 1989) or purchased from a commercial supplier, such as Clontech (Palo Alto, CA). As is appreciated by one of skill in the art of DNA amplification, care should be taken to design the primers such that the 3' ends can form a perfect hybrid with the target. A number of computer programs which assist in the design of PCR primers are available (e.g., "Oligo" primer analysis software, available from National Biosciences, Inc.).

A convenient method of performing PCR amplifications where the relative position of the peptide fragments in the protein is unknown is to use a cDNA library in a known vector as the template, employing one of the peptide specific primers in combination with a vector primer, such as a primer directed to a T3 or T7 sequences that flank the multiple cloning site in many vectors. Accordingly, if a clone containing one of the target sequences is present in the template mix, the portion between the sequence corresponding to the peptide specific primer and the end of the insert is amplified.

Additional sequences may also be obtained by hybridization screening a human DNA or cDNA library with a probe having all or a portion of the sequence represented as SEQ ID NO:5. The probe preferably contains a portion of SEQ ID NO:5 near the 5' end, to increase the chance of obtaining a clone encoding the 149 N-terminal amino acids.

In another approach, clones encoding full-length proteins may be identified in an expression library using antibodies raised against one or more of the peptides characterized by SEQ ID NOs:4 and SEQ ID NO:6. Methods for the screening of expression libraries are well-known (e.g. , Unit 6.7 of Ausubel, et al. (1988), incorporated herein by reference).

Polynucleotide sequences such as described above can be used, for example, as probes to detect other Hrs-2 sequences. Such probes typically contain at least 12, preferably at least 16, more preferably at least 20 or more consecutive nucleotides corresponding to a region contained in an Hrs-2 polynucleotide sequence.

Hrs-2 sequences may also be used to produce recombinant Hrs-2 proteins, which in turn are useful in the assays described below. Specifically, polynucleotide sequences encoding Hrs-2 proteins of the present invention may be cloned into an expression plasmid, such as p-GEX-KG, to produce corresponding polypeptides, as is described in the Examples below. Recombinant pGEX-KG plasmids can be transformed into appropriate strains of E. coli and fusion protein production can be induced by the addition of IPTG (isopropyl-thio galactopyranoside). Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using glutathione agarose affinity chromatography according to standard methods (described below; Ausubel, et al., 1988).

As described in the materials and methods, affinity chromatography may also be employed for isolating fusion proteins consisting of, e.g. , glutathione-S-transferase (GST) and the recombinant protein. In this method, detailed in the Materials and Methods under "Protein Purification", bacterial cell lysates are prepared and passed over agarose beads derivatized with glutathione as described below. This results in the attachment of the GST portions of the fusions to the glutathione on the agarose beads. The beads are then washed, and the recombinant protein is cleaved with thrombin and eluted for further analysis. It will be appreciated, however, that in some of the binding assays described below, the "immobilized" protein may be left attached to the beads for use in the assay.

Isolated recombinant polypeptides produced as described above may be further purified by standard protein purification procedures. These procedures may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.

In addition to recombinant methods, Hrs-2 proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies (as described below). Further, Hrs-2 peptides may be chemically synthesized using methods known to those skilled in the art. It will be understood that polypeptides or protein used as the "free" protein in a partner capture assay are synthesized so that they remain soluble during the binding assay. Accordingly, if SNAP-25 is used as the free protein, it may be modified for increased solubility by, for example, expressing a truncated version of the proteins that is missing its membrane anchor — i.e. , expressing only the cytoplasmic domain.

V. Screen for Modulators of Synaptic Secretion

The invention includes a method of identifying a compound capable of modulating (e.g. , potentiating or inhibiting) calcium-regulated secretion, such as the release of neurotransmitter- containing synaptic vesicles. In the method, a SNAP-25 polypeptide is contacted with an Hrs-2 polypeptide, in the presence and absence of a test compound. The effect of the test compound on the extent of binding between the SNAP-25 and the Hrs-2 polypeptides is measured, and a compound is identified as effective if its effect on the extent of binding is above a threshold level (e.g., a several- fold difference in binding level between control and experimental samples).

The test compound may be effective to enhance (potentiate) or inhibit binding between the SNAP-25 and Hrs-2 polypeptides. Compounds tested may include small molecules in a small molecule combinatorial library, peptides in a peptide combinatorial library, and the like.

It will be understood that the invention may be practiced using only a fragment of a full length Hrs-2, so long as that fragment retains the ability to bind to SNAP-25 at its normal binding site. For example, the experiments described in Examples 2, 3, and 5 were performed using the human Hrs-2 clone represented as SEQ ID NO:5. This clone is missing the N-terminal 149 amino acids of Hrs-2, yet is capable of effectively binding to SNAP-25. Accordingly, the SNAP-25 binding site (SBS) on Hrs-2 is formed by a region of Hrs-2 encoded by the partial sequence represented as SEQ ID NO: 5. Compounds which affect the binding of the SNAP-25 and Hrs-2 polypeptides to one another, when applied to and internalized by target cells, are expected to modulate vesicular release by those cells. The modulation may be an inhibition of release or stimulation of release, either when the compound is applied alone, or when the compound is applied in conjunction with another compound having an effect on vesicular release.

A. Co-Immunoprecipitation Screen In one embodiment, the assay is conducted using a co-immunoprecipitation method such as is described in the Materials and Methods and Example 2. Anti-Hrs-2 antibodies are coupled to protein A beads at a final concentration of about 2 mg/ml using the cross-linker dimethylpimelimidate (Pevsner, et al. , 1994). Rat brain membranes prepared as described herein are resuspended in 20 mM Tris, pH 8.0, 150 mM NaCl and solubilized with either 1 % CHAPS or Triton X-100 at a final protein concentration of —2 mg/ml.

The solubilized sample is incubated at 4°C for 30 min before centrifuging at 20,000 x g for 20 min. Following centrifugation, the solubilized brain membranes are pre-cleared by incubation with protein A beads (1 ml solubilized membranes per 200 μl protein A beads) for 30-60 min at 4 °C. The pre-cleared supernatant is then incubated with (i) a selected concentration of the test compound and (ii) 20 μl protein A beads with either immobilized anti-Hrs-2 antibodies or immobilized control IgG for 4-16 hrs at 4 °C. The beads are then washed three times with 300-500 μl 20 mM Tris, pH 8.0, 150 mM NaCl containing either 0.7% CHAPS or Triton X-100. Proteins bound to the beads are analyzed by Western blotting for the presence of SNAP-25 as described in the Materials and Methods and Examples below. If the test compound is effective to inhibit binding of a SNAP-25 polypeptide to the immobilized Hrs-2 polypeptide, the amount of SNAP-25 detected in the Western blot will be diminished. Conversely, if the test compound is effective to potentiate the binding of a SNAP-25 polypeptide to the immobilized Hrs-2 polypeptide, the amount of SNAP-25 detected in the Western blot will be increased. The assay is typically conducted with both negative controls (e.g. , beads coated with control rabbit Ig) and positive controls (no test compound in assays with anti-Hrs-2 antibody).

It will be appreciated that the co-immunoprecipitation assay may be practiced by using immobilized SNAP-25 and soluble Hrs-2 changing the which of using other antibodies which recognize and can immunoprecipitate the SNAP-25 polypeptide, such as anti-rsecό antibodies or antibodies directed against one or more of the other proteins in the polypeptide. B. Protein-Interaction Binding Assays

A variety of approaches may be employed to assay the binding of SNAP-25 with a corresponding SNAP-25 binding site (SBS) on Hrs-2, and/or the binding or Hrs-2 with a corresponding Hrs-2 binding site (HBS) on SNAP-25. and/or the binding of an SBS to an HBS. While the binding assays are described below using intact, full length proteins, it will be appreciated that such binding assays may be conducted using only those portions of Hrs-2 and SNAP-25 that are responsible for binding to the other. For instance, the embodiments exemplified in the Examples below were conducted using a partial-length human Hrs-2 protein (SEQ ID NO:6) which, as can be appreciated from the data, retained its ability to bind to SNAP-25. Binding assays which may be used in the practice of the invention include, but are not limited to, biochemical approaches (such as immobilized GST fusion protein constructs), bioassays (such as the yeast two-hybrid system), and physical biosensor assays (such as surface plasmon resonance). Some of these assays are described in more detail below.

1. Biochemical Assays. To identify a compound capable of affecting binding of

Hrs-2 to SNAP-25, a test compound is included in the solution (containing the "free" soluble protein; e.g., Hrs-2) that is contacted with the immobilized protein (e.g. , the GST-SNAP-25). The amount of bound Hrs-2 is detected and compared to the amount bound under similar conditions in the absence of the test compound (control). If the compound has a significant effect on the binding of Hrs-2 to the GST-SNAP-25 (i. e. , if the compound significantly increases or significantly decreases the binding), and the effect exceeds a threshold value (which is set to a desired level by the practitioner of the invention; e.g., several-fold increase or several-fold decrease in binding), the compound is identified as effective to affect or alter the binding of the Hrs-2 to SNAP-25.

Illustrations of results that may be obtained with such an assay in a multiwell plate are shown in Figs. 6 A and 6B. Both figures show fragmentary plan views of a multiwell plates. The plate 22 in Fig. 6 A is the control plate (without test compounds). Detection of binding of the free protein to the immobilized protein is indicated by the stippled pattern in the wells 24. The wells of the plate 26 in Fig. 6B each contain different test compounds, two of which inhibit the binding of the free protein to the immobilized protein. This inhibition is evidenced by a lighter stippled pattern in the wells 28 containing the effective compounds.

It will be understood that various modification of the above-described assay are included within the scope of the present invention. For example, the roles of the proteins can be switched — that is, the Hrs-2 may be immobilized to the solid support and a solution containing SNAP-25 (or a polypeptide containing the Hrs-2 binding site of SNAP-25) may be contacted with the Hrs-2. Additionally, the immobilized protein or the free protein may be exposed to a test compound prior to the binding assay, and the effects of this pre-exposure may be assessed relative to controls. Compounds identified in this manner also inhibit the binding of Hrs-2 to SNAP-25 or vice versa. Alternatively, the test compound may be added subsequent to the mixing of Hrs-2 with SNAP-25. A compound effective to reduce the level of binding in such an assay displaces Hrs-2 from SNAP-25, or vice versa.

In addition to Western blots, other, more rapid, detection schemes, such as multiwell ELISA- type approaches, may be employed. For example, a partially-purified (e.g., by the GST methods above) SNAP-25 polypeptide may be attached to the bottoms of wells in a multiwell plate (e.g., 96- well plate) by introducing a solution containing the polypeptide into the plate and allowing the polypeptide to bind to the plastic. The excess peptide-containing solution is then washed out, and a blocking solution (containing, for example, bovine serum albumin (BSA)) is introduced to block nonspecific binding sites. The plate is then washed several more times and a solution containing an Hrs-2 polypeptide and, in the case of experimental (vs. control) wells, a test compound added. Different wells may contain different test compounds, different concentrations of the same test compound, or different concentrations of Hrs-2 or SNAP-25. Further, it will be understood that various modifications to this detection scheme may be made. For example, the wells of a multiwell plate may be coated with a polypeptide containing Hrs-2, rather than SNAP-25, and binding interactions assayed upon addition of free SNAP-25. The wells may also be precoated with substance(s) that enhance attachment of the protein to be immobilized and/or decrease the level of non-specific binding. For example, the wells may be derivatized to contain glutathione and may be pre-coated with BSA, to promote attachment of the immobilized protein in a known orientation with the binding site(s) exposed.

Detection methods useful in such assays include antibody-based methods (i.e., an antibody directed against the "free" protein), direct detection of a reporter moiety incorporated into the "free" protein (such as a fluorescent label), and proximity energy transfer methods (such as a radioactive "free" protein resulting in fluorescence or scintillation of molecules incorporated into the immobilized protein or the solid support).

In particular, multiwell plates (e.g. , 96-well plates) that contain a scintillating material in the wells (available from, e.g. , Wallac, Gaithersburg, MD) may coated with the immobilized protein and used in conjunction with radioactively-labeled free protein. Free protein that binds the immobilized protein is constrained within a few nanometers of the well surface, resulting in light emission from the scintillation material in the wells. The signal can be quantitated using a plate reader or counter, such as the "MICROBETA PLUS" plate counter (Wallac), to generate standard binding plots. Such plots may be used to determine the optimal concentrations of proteins used in the assay, and may be useful in identifying compounds with more subtle effects on Hrs-2/SNAP-25 binding that can be detected using some other methods.

2. Yeast Two-Hybrid Assays. The yeast two-hybrid protein interaction assay may also be employed to identify compounds that affect the binding of Hrs-2 to SNAP-25. The assay is based on the finding that most eukaryotic transcription activators are modular (e.g, Brent, et al.,

1985), i.e., that the activators typically contain activation domains that activate transcription, and

DNA binding domains that localize the activator to the appropriate region of a DNA molecule.

In a two hybrid system, a first fusion protein contains one of a pair of interacting proteins fused to a DNA binding domain, and a second fusion protein contains the other of a pair of interacting proteins fused to a transcription activation domain. The two fusion proteins are independently expressed in the same cell, and interaction between the "interacting protein" portions of the fusions reconstitute the function of the transcription activation factor, which is detected by activation of transcription of a reporter gene. The yeast GAL4 two hybrid system (Fields and Song, 1989; Chien, et al., 1991; Durfee, et al., 1993; Bartel, et al., 1993) was developed to detect protein-protein interaction based on the reconstitution of function of GAL4, a transcriptional activator from yeast, by activation of a GAL1- lacZ reporter gene. Like several other transcription activating factors, the GAL4 protein contains two distinct domains, a DNA binding domain and a transcription activation domain. Each domain can be independently expressed as a portion of a fusion protein composed of the domain, and a second, "bait" interacting protein. The two fusion proteins are then independently expressed together in a cell. When the two GAL4 domains are brought together by a binding interaction between the two "interacting" proteins, transcription of a reporter gene under the transcriptional control of GAL4 is initiated. The reporter gene typically has a promoter containing GAL4 protein binding sites (GAL upstream activating sequences, UAS_G).

A two hybrid system such as is described above may be used to identify compounds effective to disrupt the binding of Hrs-2 to SNAP-25 as follows. A polynucleotide encoding SNAP-25 is fused to the GAL4 DNA binding domain (G4BD) in a yeast expression vector (e.g., pG4BD-SNAP-25). The vector is used to generate yeast cells harboring pG4BD-SNAP-25 and a GAL4-activated reporter gene (e.g., LacZ). These cells are then transformed with a vector carrying a fusion between the transcription activating domain of yeast GAL4 (G4AD) and Hrs-2 (e.g., pG4AD-Hrs-2). Transformants are screened (e.g., using a j3-galactosidase (0-gal) assay on plates containing the chromogenic substrate X-gal) for expression of the reporter. Reporter-expressing cells are selected, cloned, and used to screen test compounds. Compounds which increase or decrease reporter expression relative to a user-defined threshold (e.g., five-fold increase or five-fold decrease) are identified as affecting binding of the Hrs-2 to SNAP-25 and may be further evaluated, e.g., as described below, for effects on vesicular release.

C. Measuring the Effect of a Test Compound on the Extent of Binding The type of measurement used to quantify the effect of a test compound on the extent of binding between the SNAP-25 polypeptide and a Hrs-2 polypeptide depends on the type of screening assay and detection system used, and can be readily determined by one of skill in the art.

For example, in a co-immunoprecipitation screen employing a Western blot detection, such as described above, the extent of binding may be measured using densitometry of the Western blot image. The densitometry values are typically normalized, and a threshold level is set based on the amount of variation in the signal between a series of "control" samples (samples not containing test compounds). The smaller the variation, the smaller the effect of a test compound that can be reliably detected. The threshold is typically set at a several-fold difference, such as a 3-5 fold increase or decrease in binding affinity. If a multiwell plate screen is used, the output of a plate reader used to score the results of the experiment may be used as a measure of the effect on the extent of binding, and a threshold set as described above.

The precise threshold level used in a particular application of the invention is determined in view of the specific requirements of that particular application. For example, if it is desired to isolate only compounds with a very high activity, the threshold is set to a relatively high value, such as a 10 to 100-fold difference in binding affinity. If, on the other hand, it is desired to isolate compounds having a subtle effect on the binding, a lower threshold level may employed.

It is also appreciated that, as with any other compound screening assay, the effect of a particular compound on the binding of a SNAP-25 polypeptide to an Hrs-2 polypeptide depends on the concentration of the compound. At relatively high compound concentrations the effect may be large, and be manifested as, e.g., a 50-fold difference in binding between control and experimental samples, whereas at lower compound concentrations, the effect may be smaller.

D. Effects of Identified Compounds on Vesicular Release Compounds identified by a screen such as one described above as affecting the binding of an

SNAP-25 polypeptide to an Hrs-2 polypeptide may be further evaluated for their ability to modulate vesicular release in vitro and in vivo.

For example, the compounds may be tested using the PC 12 cell Dj3H vesicular release assay (Bennett, et al. , 1993), which detects a membrane-associated form of the enzyme dopamine β- hydroxylase (D3H) on the luminal side of catecholamine-containing granules. When the cells are depolarized in the presence of calcium, granule fusion with the plasma membrane results in the exposure of D/3H on the cell surface, where it can be quantitatively detected by immunofluorescence microscopy (Elferink, et al. , 1983). By treating a sample of cells with a compound identified as affecting the binding of a SNAP-25 polypeptide to an Hrs-2 polypeptide, depolarizing the cells (e.g., with a pulse of KC1) in the presence of calcium, and comparing the response to that obtained with an untreated sample of cells, the effects of the compound on vesicle release in PC 12 cells may be assessed. Similar assays may be employed using freshly-isolated cells (e.g., in brain slices), or suitable animal models.

Since the interactions between Hrs-2 and SNAP-25 that affect vesicular release appear to be primarily intracellular in nature, the compounds will likely need to be internalized by the target cells to have the desired effect on vesicle release. Methods of promoting uptake of different types of compounds by cells are well known in the art. For example, certain classes of compounds, e.g. , lipophilic compounds and esters, can simply diffuse across the lipid bilayer. Other types of compounds may utilize membrane transport proteins to be internalized, and still others can be internalized by endocytosis or liposome-mediated targeting.

VI. Compounds Suitable for Screening

A variety of different compounds may be screened using methods of the present invention. They include peptides, macromolecules, small molecules, chemical and/or biological mixtures, and fungal, bacterial, or algal extracts. Such compounds, or molecules, may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.

A set of potentially-effective test peptides can be generated from overlapping peptides spanning the entire sequence of each of the proteins involved in the SNAP-25 polypeptide/Hrs-2 polypeptide interaction. Such a set is likely to contain peptides which may be effective to disrupt the interactions of the SNAP-25 polypeptide with the Hrs-2 polypeptide.

In cases where an identified active compound is a peptide. the peptide may be utilized to aid in the discovery of orally-active small molecule mimetics.

VII. Applications

Inhibitory compounds isolated using methods of the present invention may be employed to inhibit or enhance vesicle-mediated secretion from cells. Similarly, compounds which enhance or potentiate the binding of an SNAP-25 polypeptide to an Hrs-2 polypeptide may be used to upregulate vesicle-mediated secretion. The ability to modulate secretion processes has utility in a variety of areas, some of which are identified below.

A. CNS Disease Applications A number of disorders and/or conditions of the central nervous system (CNS) may be alleviated by selectively enhancing or inhibiting vesicular release in specific areas of the brain. They include affective disorders (e.g., depression), disorders of thought (e.g., schizophrenia) and degenerative disorders (e.g. , Parkinson's disease), as well as applications such as anesthesia. A variety of drugs are currently used to treat such disorders and/or conditions. Compounds identified by methods of the present invention may be used either alone, or in combination with currently used therapies to alleviate symptoms associated with the disorders.

Drugs used to treat affective disorders, which include depression, manic-depressive disorders and anxiety disorders, typically fall into three classes: (i) monoamine oxidase (MAO) inhibitors, such as phenelzine, (ii) tricyclic compounds, such as imipramine, and (iii) serotonin uptake blockers, such as fluoxetine and trazodone. All of these drugs work, at least in part, by increasing the concentration of either serotonin or biogenic amine neurotransmitters in CNS synapses of treated individuals. According to methods of the present invention, compounds which enhance the release of serotonin or biogenic amines at selected brain synapses may be similarly effective at treating depressive disorders. Such compounds may be identified by screening for compounds effective to enhance the binding of Hrs-2 to SNAP-25.

Disorders of thought, such as schizophrenia, have been treated with a variety of antipsychotic drugs (including phenothiazines, such as chlorpromazine, butyrphenones, such as haloperidol, xithioxanthenes, and newer drugs, such as clozapine) now known to act as blockers of dopamine receptors. According to the teachings presented herein, compounds identified as inhibitors of release of dopamine-containing vesicles, particularly vesicles released from cells having their cell bodies in the arcuate nucleus of the hypothalamus, the substantia nigra, or the ventral tegmental area, may be employed to relieve symptoms of schizophrenia.

Neurodegenerative diseases, such as Parkinson's disease and Huntington's disease, may also benefit from compounds identified according to the methods of the present invention. Parkinson's disease is due to degeneration of the nigrostriatal pathway, raphaei nuclei, locus ceruleus, and motor nucleus of vagus, which result in a reduction of dopamine, serotonin and norepinephrine levels. The symptoms of Parkinson's may be alleviated by administering compounds identified according to the teachings presented herein as stimulating release of vesicles containing the above neurotransmitters.

B. Other Applications In addition to applications in the CNS, compounds identified employing methods of the present invention may be used to therapeutically intervene in a variety of other systems. They include the endocrine system for treatment of hormonal imbalances, the immune system for intervention in antigen processing, secreted immunomodulators, and viral processing, as well as anti-tumor applications, such as regulation of membrane trafficking during rapid cell division.

The following examples illustrate but in no way are intended to limit the present invention.

Materials and Methods Unless otherwise indicated, the following sources were used for reagents and materials:

[¹²⁵I]goat anti-rabbit secondary antisera and the Enhanced Chemo-Luminescence (ECL) system were obtained from Amersham Corp. (Arlington Heights, IL). Nitrocellulose paper was obtained from Schleicher and Schuell (Keene, NH). Materials for SDS-polyacrylamide gel electrophoresis (SDS- PAGE) were obtained from Bio-Rad Laboratories (Hercules, CA). Other chemicals were purchased from Sigma (St. Louis, MO) or United States Biochemical (Cleveland, OH). All protein purification procedures were carried out at 4°C unless otherwise noted.

A. Buffers

Phosphate-buffered saline (PBS) lOx stock solution, 1 liter:

80 g NaCl

2 g KCl

11.5 g Na,HP04-7HX>

2 g KH₂P0₄ Working solution, pH 7.3:

137 mM NaCl

2.7 mM KC1

4.3 mM Na₂HP0₄-7H₂0

1.4 mM KH 0₄

Buffer A

PBS

0.05 % "TWEEN 20"

2 mM EDTA 0.1 % /3-mercaptoethanol

1 mM phenylmethylsulfonyl fluoride (PMSF) 5 μg/ml apoprotein

3 μg/ml pepstatin 3 μg/ml leupeptin

Buffer B

0.32 M sucrose

10 mM Hepes, pH 7.5 5 μg/ml apoprotein 3 μg/ml pepstatin 3 μg/ml leupeptin Cleavage Buffer

50 mM Tris-HCl (pH 8.0) 150 mM NaCl 2.5 mM CaCl₂ 0.1 % /3-mercaptoethanol 10 μg/ml thrombin ( ~ 3000 U/mg)

B. Two-hybrid Screen and Cloning

The entire coding region of mouse SNAP-25b (Oyler, et al. , 1989) was amplified using the polymerase chain reaction using primers S25F (SEQ ID NO:l - forward: 5' CCG AAT TCA TGG CCG AGG ACG CAG ACA 3') and S25R (SEQ ID NO:2 - reverse: 5' CCG TCG ACT AAC CAC TTC CCA GCA TCT 3') containing internal EcoRI and Sail restriction sites. Amplification products were inserted in frame with the GAL4 binding domain (GAL4BD) in the pGBT9 vector (Fields and Song, 1989) creating PGBT9/SNAP-25. All constructions were verified by sequencing (Sequenase, USB). A human brain cDNA library inserted downstream of the GAL4 activation domain (GAL4AD) in the pGADIO vector was purchased from Clontech (Palo Alto, CA). Yeast (HF7c strain, Clontech) were cotransformed (Gietz and Schiestl, 1991) with the pGAD/library and pGBT9/SNAP-25 (4 x 10⁶ colonies), plated on agar containing yeast nitrogen base (6.7 g/L, Difco Laboratories, Detroit, MI), adenine (0.054 mM), lysine (0.165 mM), dextrose (2%) and 3-amino-l,2,4-triazole (10 mM, Sigma) and stored at 30°C in the dark.

On the 16 positive clones recovered, multiple cotransformations were performed with the resultant candidate DNA and the pGBT9 plasmid alone, pGBT9/SNAP-25, and pGBT9/lamin. Clones that reacted positively for 0-galactosidase activity with the pGBT9/SNAP-25 but not either by themselves or with the control plasmids were considered for further study. A rat brain library (λ ZAP II, Stratagene) was screened with a random primed ³²P-labeled insert from a human Hrs-2 clone, enabling the isolation of a single clone contained the full coding region including a consensus translational start site (Kozak, 1986). An additional clone included an in-frame stop codon upstream of the first methionine confirming its position.

C. Protein Purification

Glutathione-S-transferase (GST) fusion proteins of Hrs-2 (GST-Hrs-2) were prepared using previously described methods (Pevsner et al., 1994). Briefly, the partial human Hrs coding sequence (SEQ ID NO: 5), isolated as described above, was cloned into the pGEX-derived (Smith and Johnson, 1988) vector, pGEX-KG (Guan and Dixon, 1991). The resultant vectors were used to transform XL-1 Blue E. coli cells (Stratagene, La Jolla, CA). Bacterial clones containing the protein sequences were selected and grown at 37°C, with vigorous agitation, for approximately 4 hours in 1-liter of liquid culture (Luria Broth (LB); Howard Hughes Media Supply Facility, Stanford University, Stanford, CA). 1 ml of 100 mM isopropyl-l-thio-/3-D-galactoside (IPTG) was added to induce protein expression, and the culture was incubated for approximately another three hours.

The cells were pelleted and resuspended in 10 ml ice-cold Buffer A, lysed with a French Press (SLM Aminco, Rochester, NY) until translucent, centrifuged briefly to pellet cellular debris, and the supernatant transferred to a fresh tube. The fusion protein was purified by affinity chromatography as follows. Five ml of a 50%

(v/v) slurry of pre-swelled glutathione-agarose beads were added to the supernatant and mixed gently for approximately 1 hour at room temperature to allow fusion protein in the supernatant to bind to the beads. The beads were then washed three times to remove any unbound protein. Each wash consisted of adding 10 ml PBS, mixing, and centrifuging in a table-top centrifuge for ~ 5 minutes at maximum speed (2000 x g) to collect the beads.

The fusion protein was typically eluted from the beads using the thrombin cleavage protocol

(Ausubel, et al. , 1988). Briefly, 10-20 ml of the bead slurry were combined with 10 ml Cleavage

Buffer and incubated at 25 °C for about 1 hour. Phenylmethylsulfonyl fluoride (0.6 mM final concentration) was then added to the protein elution, and the sample was concentrated to 0.5 ml using a "CENTRIPREP" concentrator (Amicon Inc., Beverly, MA).

The Hrs-2 protein was then size fractionated by gel filtration on a "SUPEROSE 12" sizing column (Pharmacia, Piscataway, NJ) using a buffer consisting of 50 mM Tris, 500 mM NaCl, and 0.05 % "TWEEN 20" .

His-tagged Hrs-2 was purified using methods described previously (Sollner, et al., 1993b). Briefly, the coding region of the Hrs-2 sequence was cloned into the £røRI site of pRSET-B (Invitrogen, Carlsbad, CA). The resulting plasmid and pREP4 (Qiagen, Chatsworth, CA) were transformed into E. coli (HB101, Stratagene, La Jolla, CA).

Transformed cells were grown to a density of about A₆₆₀=0.8 in LB (Howard Hughes Media Preparation Facility, Stanford, CA) supplemented with 100 μg/ml ampicillin and 125 μg/ml kanamycin. Protein synthesis was induced by treating the cells for 3 hours with 1 mM IPTG.

His₆-Hrs-2-expressing cells were collected, washed in Buffer A, disrupted in a French Press, and the suspension was clarified by centrifugation at 100,000 Xg for 1 hour. The supernatant was passed over a Ni-NTA-agarose column (Qiagen; 5 ml bed volume) and the His₆-Hrs-2 was eluted with a two step imidazole wash -50 mM followed by 500 mM imidazole. Protein concentrations for the above-isolated proteins were estimated by Coomassie blue staining of protein bands after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) using bovine serum albumin (BSA) as a standard.

D. Preparation of Anti-Hrs-2 Antisera

Anti-Hrs-2 antisera were prepared in rabbits with gel-purified recombinant Hrs-2 protein, prepared as described above, using standard methods (Harlow, et al., 1988). Briefly, -250 μg of protein isolated from a polyacrylamide gel was suspended in — 1 ml PBS and injected subcutaneously (sc) once every three weeks for 12 weeks. The initial injection contained complete Freund's adjuvant, while subsequent injections contained incomplete Freund's adjuvant. Serum was isolated after 12 weeks and used as described below.

E. NTPase Assay

NTPase reactions (lOOμl) contained Hrs-2 (10μl of Superose 12 column fractions pooled from monomer and oligomer peaks), unlabeled NTPs at various concentrations, 1.5-2.0 μCi of the corresponding [γ-³²P] NTPs, 1 mM MgCl₂, 0.1 mM EDTA, 0.2% (w/v) BSA, and 50 mM Tris-HCl, pH 7.4. Reactions were conducted for 20 min at 25 °C and were terminated by addition of an ice-cold slurry containing 5% (w/v) activated charcoal and 50 mM NaH₂P0₄, pH 2.0. Radioactivity in supernatants of reaction mixtures was determined by scintillation counting. Blank values were determined using column elution buffer in the reaction mixture. Steady state NTPase activity of Hrs-2 is expressed as molar turnover number (moles of NTP hydrolyzed per mol Hrs-2 per minute). Data shown are the meaα+S.D. of one representative experiment performed in triplicate. Very similar results were obtained in two-three independent experiments with different preparations of Hrs-2.

F. Binding

For binding experiments, either increasing concentrations (0.11-7 μM) of Hrs-2 were incubated with immobilized GST-SNAP-25 (0.3 μM, 4°C for 60 min) in the presence and absence of Zn²⁺ (1 mM) or to immobilized GST (X), or a single concentration of Hrs-2 (2 μM) was incubated with immobilized GST-SNAP-25 in the presence of various free calcium concentrations. After washing, the beads were boiled in SDS-containing sample buffer and proteins were separated by SDS- PAGE. After transfer to nitrocellulose, blots were probed with Hrs-2 antibodies and ¹²⁵I-labeled secondary antisera. Hrs-2 was visualized/quantitated using a phosphorimager. G. Immunoprecipitation

Affinity-purified anti-Hrs-2 antiserum or rabbit IgG were covalently bound to protein A- sepharose beads at a final concentration of 2 mg/ml with dimethylpimelimidate for 30 minutes at 4°C, essentially as previously described (Pevsner, et al. , 1994). Rat brain post-nuclear supernatant was prepared as follows. Frozen rat brains were homogenized in 20 ml of Buffer B with a Teflon-glass homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The pellet was dissolved in 1 ml 20 mM Tris, pH 8.0 and 150 mM NaCl and solubilized with 1 % Triton X-100 at a final protein concentration of — 2 mg/ml.

The solubilized sample was incubated at 4 °C for 30 min before centrifuging at 20,000 x g for 20 min. Following centrifugation, the solubilized brain membranes were pre-cleared by incubation with protein A beads (1 ml solubilized membranes per 200 μl protein A beads) for 60 min at 4 °C.

Immunoprecipitation was performed by incubating the detergent solubilized rat brain post nuclear supernatant with the antibody-conjugated beads for 16 hours at 4°C. The beads were then washed three times with 500 μl 20 mM Tris, pH 8.0, 150 mM NaCl containing 0.7% Triton X-100 and resuspended in 30 μl of sample buffer. The sample was separated by SDS-PAGE (10%), transferred to nitrocellulose, and probed with antibodies. Proteins were visualized using chemiluminescence (Amersham ECL).

H. Permeabilized Cell Secretion Assay PC12 cells were maintained as described (Lomneth, et al., 1991) and secretion was assayed essentially as described (Hay and Martin, 1992, incoφorated herein by reference). Exogenous protein was added into the priming (and removed prior to triggering) or triggering reactions to examine its effect on each secretion step.

A nonspecific effects of the high molecular weight control protein (α-cat) was observed when it was added in equivalent concentrations. GST protein had no effect on secretion at molar concentrations 4 times the highest concentration of Hrs-2 tested. Six secretion experiments were performed using either 6XHIS-Hrs-2 (3) or GST-Hrs-2 (3).

Dose response curves to ATP in the presence and absence of Hrs-2 revealed an inability of increased ATP to overcome the inhibition of secretion by Hrs-2, suggesting that the inhibition of secretion is not simply due to degradation of necessary ATP by Hrs-2. Each condition was tested in duplicate. EXAMPLE 1 Cloning and Sequence Analysis of Hrs-2 A human cDNA encoding a protein that interacts selectively with SNAP-25 was isolated as described in the materials and methods using the yeast two hybrid approach (Fields and Song, 1989). The putative protein encoded by a corresponding rat clone is similar to Hrs, a growth factor-induced phosphoprotein (Komada and Kitamura, 1995). The novel proteins described herein were therefore termed "Hrs-2". However, the presently-described Hrs-2 sequences suggest that the previously- published sequence of Hrs is either incorrect or is a splice variant of Hrs-2 in the carboxy terminus. As detailed in the Materials and Methods, both a rat and a human clone were identified. The coding sequence of the rat clone encodes a 925 amino acid peptide with a predicted molecular mass of 106,764 kDa. The nucleotide sequence encoding rat Hrs-2 is presented herein as SEQ ID NO:3, with the amino acid sequence represented as SEQ ID NO:4. The human clone initially recovered in the two hybrid screen contained all of the rat clone except the amino terminal 149 amino acids. The nucleotide sequence of the human clone is presented as SEQ ID NO:5; the amino acid sequence as SEQ ID NO:6.

Figure 1 is a schematic illustration of the domain structure of Hrs-2. The predicted ORF from the rat cDNA contains domains with potential functional significance. A zinc finger (Zn finger) composed of 8 cysteine residues is present in the amino terminal half of the protein. This region surrounding the 8 cysteine residues that form two zinc coordination domains (Zn²⁺ finger) is well conserved among a variety of proteins suggested to be involved in protein trafficking to the yeast vacuole (e.g. Fabl, VPS 27, Vac lp) or to the endosome in eukaryotic cells (e.g. pl62).

A putative nucleotide binding site consisting of GXXXXGK (SEQ ID NO:7), RDET (SEQ ID NO:8), DXXG (SEQ ID NO:9), and TQKXD (SEQ ID NO: 10) is also present (Bourne, et al. , 1991). The first three domains of the putative nucleotide binding site are identical to the consensus (G1,G2,G3) while the fourth domain (G4) is a variant found in some GTP-binding proteins (Bourne, et al., 1991). The G4 domain is thought to confer selectivity for the nucleotide although some proteins specific for GTP completely lack the G4 domain (Bourne, et al. , 1991; Dever, et al. , 1987; Cheng, et al. , 1991). The spacing between the domains appears larger than usual and it is not yet clear whether the putative G4 domain we identified functions to coordinate nucleotide binding (Dever, et al., 1987; Cheng, et al. , 1991).

The Hrs-2 protein sequence also possesses two 28 amino acid regions with the potential (p=0.74 and p=0.97, respectively) to form coiled-coil structure (indicated as "coiled-coil"), as well as a glutamine and proline rich region ("Q/P rich region").

These data suggest a role for Zn²⁺ ions, nucleotides, and coiled-coil mediated protein-protein interactions in Hrs-2. EXAMPLE 2 Characterization and Expression Pattern of Hrs-2 The expression of Hrs-2 was evaluated by immunoblot analysis of various rat tissues (indicated) using a polyclonal antibody raised against Hrs-2 bacterial fusion protein. Immunoblot analysis revealed a single band of approximately 115 kDa, with the greatest abundance in brain. A Western blot experiment was also conducted with rat brain post nuclear supernatant (PNS) using SNAP-25 antisera. Taken together, the results indicate that brain postnuclear supernatant contains both Hrs-2 and SNAP-25.

The Hrs-2 antiserum was also used to perform immunoprecipitation studies from rat brain PNS. These experiments showed that both Hrs-2 and SNAP-25 are present in the rat brain PNS sample. Anti-Hrs-2 antisera (αHrs-2), but not rabbit IgG, resulted in precipitation of both Hrs-2 and SNAP-25. These results provide evidence for the interaction of Hrs-2 and SNAP-25 in vivo.

Direct and saturable binding of Hrs-2 to GST-SNAP-25 immobilized on glutathione-agarose beads was evaluated in the presence and absence of Zn²⁺ as described in the Materials and Methods (preliminary experiments showed that recombinant Hrs-2 is able to bind Zn²⁺ ions). The results are shown in Figure 2A. Data quantitated from autogradiographs are plotted in Fig. 2A. Pixel values

(X 1000) for Hrs-2 were: 0.125μM, 238; 0.25μM, 767; 0.5μM, 1652; lμM, 5724; 2μM, 7728; 4μM,

8677; 7μM, 9012. Pixel values (X 1000) for Hrs-2 + Zn²⁺ were: 14, 20, 187, 413, 847, 1422, 1772.

The stoichiometry of the interaction between Hrs-2 and SNAP-25 was calculated to be in the range of 0.1-0.55: 1 (Hrs-2: SNAP-25). The stoichiometry was determined by quantifying the amount of Hrs-2 bound to SNAP-25 when one of the proteins was immobilized on glutathione agarose beads and the other was soluble. The range was due to slightly different results obtained depending on whether Hrs-2 or SNAP25 was immobilized.

The results demonstrate that although the binding of Hrs-2 to SNAP-25 remained saturable in the presence of Zn²⁺, the maximum binding was reduced about four-fold. The inhibition of Hrs-2- SNAP-25 binding by Zn²⁺ suggests that the inhibition of Hrs-2-SNAP-25 binding may facilitate transmitter release.

In addition to Zn²⁺ ions, the zinc finger or other domains of Hrs-2 may bind calcium.

Accordingly, experiments were performed to determine the extent Hrs-2 binding to immobilized GST- SNAP-25 in the presence and absence of increasing concentrations of free calcium. The results are shown in Fig. 2B. Pixel values (X 10,000) for Hrs-2 were: OμM (-EGTA), 20.13; OμM, 20.32; 3μM,

20.31; 30μM, 11.95; 100μM, 9.713; 300μM, 7.974; 1 mM, 5.80; 3 mM, 4.472; 10 mM, 3.574.

The results show that the binding of Hrs-2 to GST-SNAP-25 was not different in the absence or presence of EGTA (2 mM). As the concentration of free calcium was increased the apparent binding of Hrs-2 to SNAP-25 decreased. Barium (1 mM) weakly inhibited (was ~ 100 times less effective than calcium) while strontium (1 mM) had no effect on the apparent binding of Hrs-2 to GST-SNAP-25. The results show that the Hrs-2/SNAP-25 interaction is altered by calcium in the physiological concentration range that has been shown to support regulated secretion (Smith and Augustine, 1988).

EXAMPLE 3 Nucleotide Binding and Hydrolysis Activities of Hrs-2 Since the Hrs-2 primary structure contains putative nucleotide binding motifs, the ability of recombinant Hrs-2 to bind and hydrolyze nucleotides was examined as described in the Materials and Methods. The results are shown in Figures 3A-E.

GST-tagged Hrs-2 was purified by affinity chromatography and gel filtration. Fig. 3A shows that recombinant Hrs-2 eluted from the gel filtration column in two peaks. The top of the figure shows the profile of fractions of eluate probed with the anti-Hrs-2 antibody, the bottom part shows the protein elution profile (A₂₈₀) from the gel filtration column. The position of one peak was consistent with monomeric protein while the other peak migrated at a position consistent with a multimer.

The ATPase, GTPase, and UTPase activities contained in the peaks were analyzed as described in the Materials and Methods. The results are shown in Figs. 3B and 3C. Fig. 3B shows the NTPase activity of monomeric Hrs-2 (right column peak in Fig. 3A), while Fig. 3C shows the NTPase activity of oligomeric Hrs-2 (left column peak in A). The symbols in both figures are as follows: [■], ATPase; [ A] , GTPase; [▼], UTPase. The results show that both peaks possess ATPase, GTPase, and UTPase activities.

The substrate-response curves for monomeric Hrs-2 ATPase, GTPase, and UTPase activity were best fit by single site models (r²=0.99, 0.99, and 0.98, respectively) with apparent K„ of 152μM, 123μM, and 77μM and K^ of 0.41, 0.18, and 0.46 min^"1 respectively (Figure 3B). The nucleotidase activity of the oligomeric form of Hrs-2 demonstrates that ATP is the preferred substrate. The kinetic data for the oligomer was also best fit by single site models (r²⁼0.99, 0.99, 0.95, respectively) with apparent K^ of 53μM, 487μM, and 61μM and K_cal of 2.64, 2.08, and 0.32 min^"1 respectively (Figure 3C). These kinetic studies, and the fact that intracellular concentrations of ATP are higher than GTP and UTP, suggest that under physiological conditions ATP is the nucleotide triphosphate substrate for the Hrs-2 enzyme, and that the most active form of the enzyme is an oligomer. The K^ and K,._at values of Hrs-2 for ATP are similar to what has been previously reported for NSF, another oligomeric ATPase that physically interacts with other components of the secretory apparatus (Whiteheart, et al. , 1994; Tagaya, et al. , 1993; Morgan, et al. , 1994). Additional experiments were performed to assess the effects non-hydrolyzable nucleotide analogs on the ATPase activity of monomeric Hrs-2. ATPase activity was assessed in the presence of lμM ATP with various concentrations of ATP7S and GTPγS. The results are shown in Figs. 3D

(ATP7S) and 3E (GTP7S). The IC₅₀ values for ATP7S and GTPγS were 815nM and 843nM, respectively.

The results show that both ATP7S and GTPγS inhibited the ATPase activity of monomeric

Hrs-2 with similar potency and in a monophasic manner, indicating the presence of a single nucleotide binding site. This is consistent with the proposed single nucleotide binding site in the deduced amino acid sequence. The ATPase activity of the oligomeric form was also inhibited by ATP7S and GTP7S, as well as EDTA, suggesting that Hrs-2 requires Mg²⁺ for this enzyme activity.

EXAMPLE 4 Immunohistochemical Localization of Hrs-2 Primary hippocampal CA3/CA1 cultures were obtained and maintained as described by Malgaroli and Tsien, 1992. Immunocytochemistry was carried out as previously described (Ting, et al., 1995) using affinity-purified anti-Hrs-2 rabbit polyclonal antibodies at 1:400 dilution as primary antibodies. The labeling was visualized using DTAF-labelled donkey anti-rabbit or Texas Red-labeled sheep anti-mouse antibodies.

The results are shown in Figs. 4A-4D. Fig. 4 A shows hippocampal neurons immunolabeled with anti-Hrs-2 antibodies and stained with secondary antibodies conjugated to DTAF. Fig. 4B shows hippocampal neurons immunolabeled with anti-synaptophysin antibodies and stained with secondary antibodies conjugated to Texas Red. Fig. 4C shows hippocampal neurons immunolabeled with anti- Hrs-2 antibodies and stained with secondary antibodies conjugated to DTAF. Fig. 4D shows hippocampal neurons immunolabeled with anti-SNAP-25 antibodies and stained with secondary antibodies conjugated to Texas Red. Arrows point to some regions of overlapping signal. The size bar (= 10 μm) is the same for all panels.

The staining in the cultured hippocampal cells revealed a cytosolic localization of Hrs-2 that included punctate structures (Figure 4 A and C). Comparison of the Hrs-2 labeling pattern with that obtained with antibodies against synaptophysin (Figure 4B) and SNAP-25 (Figure 4D) revealed considerable coincidence. Thus, the Hrs-2 protein is most abundantly expressed in brain (Figure IB) where apparently colocalizes with SNAP-25 and synaptic vesicles in axons and nerve terminals, consistent with its role in transmitter secretion. EXAMPLE 5 Inhibition of Exocytosis by Hrs-2 To test whether Hrs-2 modulates exocytosis, recombinant Hrs-2 was added to a permeabilized PC 12 cell reconstitution system for MgATP-dependent, calcium-regulated, ³H-norepinephrine (NE) secretion (Hay and Martin, 1992), as described in the Materials and Methods.

The results are shown in Figure 5. Secretion is presented as the percentage of [³H]-NE released of the total [³H] added in each individual triggering reaction ([³H] in supernatant / ([³H ] in supernatant + [³H] in pellet)) compared to the maximum stimulated secretion produced when adding all reaction components. Symbols: Hrs-2 (D), GST (■), GST- catenin (T), GST-SNAP-25 (•), and GST-α-SNAP (v).

The results show that Hrs-2 dose-dependently and saturably inhibits the release of ³H- norepinephrine (NE) from permeabilized PC12 cells. Four control proteins, glutathione-S-transferase (GST), GST-SNAP-25, GST-alpha-SNAP (Bennett and Scheller, 1994; Scheller, 1995; Sϋdhof, 1995; Rothman, 1994) and GST-α catenin (a 128 kD protein that is not involved in neurotransmission but implicated in cell-cell adhesion) (Kemler, 1993), were without effect on ³H-NE secretion at molar concentrations equal to or greater than those at which Hrs-2 inhibited secretion. Recombinant syntaxin (cytoplasmic domain) inhibited secretion with efficacy similar to that of Hrs-2 (Figure 5).

These data provide a functional demonstration of Hrs's ability to regulate the secretory apparatus.

While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

(i) APPLICANT: Bean, Andrew J.

Scheller, Richard H.

(ii) TITLE OF INVENTION: Methods and Compositions for Modulation of Vesicular Release

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Dehlinger & Associates

(B) STREET: 350 Cambridge Avenue, Suite 250

(C) CITY: Palo Alto

(D) STATE: CA

(E) COUNTRY: USA (F) ZIP: 94306

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS -DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE: 26-FEB-1997

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Sholtz, Charles K. (B) REGISTRATION NUMBER: 38,615

(C) REFERENCE/DOCKET NUMBER: 8600-0183

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 324-0880 (B) TELEFAX: (415) 324-0960

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Primer S25F

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

CCGAATTCAT GGCCGAGGAC GCAGACA 27

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Primer S25R

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CCGTCGACTA ACCACTTCCC AGCATCT 27

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2817 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Rat Hrs-2 cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 43..2814

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GTTTGAGCCC GCGGCACGAG GTCGGTTCGG AGTGGGGTCG CC ATG GGG CGA GGC 54

Met Gly Arg Gly

1 AGC GGC ACC TTC GAG CGT CTC CTA GAC AAA GCC ACC AGC CAG CTT CTA 102 Ser Gly Thr Phe Glu Arg Leu Leu Asp Lys Ala Thr Ser Gin Leu Leu 5 10 15 20

TTG GAG ACA GAC TGG GAG TCC ATT CTA CAG ATC TGC GAC CTG ATC CGT 150 Leu Glu Thr Asp Trp Glu Ser lie Leu Gin lie Cys Asp Leu lie Arg

25 30 35

CAG GGG GAC ACA CAA GCA AAA TAT GCT GTA AAC TCC ATC AAG AAG AAG 198 Gin Gly Asp Thr Gin Ala Lys Tyr Ala Val Asn Ser lie Lys Lys Lys 40 45 50

GTC AAT GAT AAG AAC CCA CAT GTG GCT TTG TAT GCT CTG GAG GTG ATG 246 Val Asn Asp Lys Asn Pro His Val Ala Leu Tyr Ala Leu Glu Val Met 55 60 65

GAG TCT GTG GTA AAG AAC TGT GGC CAG ACA GTC CAT GAT GAA GTG GCC 294 Glu Ser Val Val Lys Asn Cys Gly Gin Thr Val His Asp Glu Val Ala 70 75 80

AAC AAA CAG ACC ATG GAA GAA CTG AAG GAG CTG CTG AAG AGG CAA GTG 342 Asn Lys Gin Thr Met Glu Glu Leu Lys Glu Leu Leu Lys Arg Gin Val 85 90 95 100

GAA GTT AAT GTT CGG AAC AAG ATC TTG TAC CTG ATC CAG GCC TGG GCA 390 Glu Val Asn Val Arg Asn Lys lie Leu Tyr Leu lie Gin Ala Trp Ala 105 110 115

CAT GCC TTC CGG AAT GAA CCC AAG TAC AAG GTG GTC CAG GAC ACA TAC 438 His Ala Phe Arg Asn Glu Pro Lys Tyr Lys Val Val Gin Asp Thr Tyr 120 125 130 CAG ATC ATG AAG GTA GAA GGA CAT GTC TTC CCT GAG TTT AAG GAG AGC 486 Gin lie Met Lys Val Glu Gly His Val Phe Pro Glu Phe Lys Glu Ser 135 140 145

GAC GCC ATG TTC GCT GCT GAA AGA GCC CCT GAC TGG GTG GAT GCT GAG 534 Asp Ala Met Phe Ala Ala Glu Arg Ala Pro Asp Trp Val Asp Ala Glu 150 155 160

GAA TGC CAT CGG TGC AGA GTA CAG TTT GGA GTG GTG ACC CGC AAG CAT 582 Glu Cys His Arg Cys Arg Val Gin Phe Gly Val Val Thr Arg Lys His 165 170 175 180

CAC TGC CGA GCG TGT GGG CAG ATC TTT TGT GGC AAG TGT TCC TCC AAG 630 His Cys Arg Ala Cys Gly Gin lie Phe Cys Gly Lys Cys Ser Ser Lys 185 190 195

TAC TCC ACC ATC CCC AAG TTC GGC ATT GAG AAG GAA GTG CGC GTG TGT 678 Tyr Ser Thr lie Pro Lys Phe Gly lie Glu Lys Glu Val Arg Val Cys 200 205 210 GAG CCC TGC TAT GAG CAG CTG AAC AAG AAG GCA GAA GGG AAA GCT GCC 726 Glu Pro Cys Tyr Glu Gin Leu Asn Lys Lys Ala Glu Gly Lys Ala Ala 215 220 225

TCT ACC ACT GAG CTG CCC CCA GAG TAC CTG ACC AGC CCC CTG TCA CAG 774 Ser Thr Thr Glu Leu Pro Pro Glu Tyr Leu Thr Ser Pro Leu Ser Gin 230 235 240

CAG TCT CAG CTG CCC CCA AAG CGG GAT GAG ACA TGC GCT GCA GGA AGA 822 Gin Ser Gin Leu Pro Pro Lys Arg Asp Glu Thr Cys Ala Ala Gly Arg 245 250 255 260

GGA GGA GCT ACA GCT GGC TCT GGC CAT TCA CAG TCA GAG GCT GAG GAG 870 Gly Gly Ala Thr Ala Gly Ser Gly His Ser Gin Ser Glu Ala Glu Glu 265 270 275

AAG GAA AGG ATG AGA CAG AAG TCA ACA TAC ACA GCG CAT CCA AAG TCA 918 Lys Glu Arg Met Arg Gin Lys Ser Thr Tyr Thr Ala His Pro Lys Ser 280 285 290 GAG CCT GCG CCC TTG GCT TCC TCT GCA CCC CCA GCT GGT AGC CTG TAC 966 Glu Pro Ala Pro Leu Ala Ser Ser Ala Pro Pro Ala Gly Ser Leu Tyr 295 300 305

TCC TCG CCT GTG AAC TCA TCA GCA CCT CTG GCT GAG GAC ATC GAC CCT 1014 Ser Ser Pro Val Asn Ser Ser Ala Pro Leu Ala Glu Asp lie Asp Pro 310 315 320

GAG CTT GCA AGA TAC CTC AAC CGG AAC TAC TGG GAG AAG AAA CAG GAA 1062 Glu Leu Ala Arg Tyr Leu Asn Arg Asn Tyr Trp Glu Lys Lys Gin Glu 325 330 335 340

GAA GCT CGG AAG AGC CCC ACA CCA TCT GCA CCT GTG CCC CTG ACC GAG 1110 Glu Ala Arg Lys Ser Pro Thr Pro Ser Ala Pro Val Pro Leu Thr Glu 345 350 355

CCA GCT GCC CAG CCC GGA GAA GGA CAT ACA GCC CCC AAC AGC ATG GTA 1158 Pro Ala Ala Gin Pro Gly Glu Gly His Thr Ala Pro Asn Ser Met Val 360 365 370

GAG GCC CCT CTT CCA GAG ACA GAC TCT CAG CCC ATA ACT TCC TGC AGT 1206 Glu Ala Pro Leu Pro Glu Thr Asp Ser Gin Pro lie Thr Ser Cys Ser 375 380 385

GGC CCC TTT AGT GAG TAC CAG AAC GGG GAG TCG GAG GAG AGC CAC GAG 1254 Gly Pro Phe Ser Glu Tyr Gin Asn Gly Glu Ser Glu Glu Ser His Glu 390 395 400

CAG TTC CTC AAG GCC CTG CAG AAT GCA GTC AGC ACT TTT GTC AAC CGC 1302 Gin Phe Leu Lys Ala Leu Gin Asn Ala Val Ser Thr Phe Val Asn Arg 405 410 415 420 ATG AAG AGC AAC CAC ATG AGG GGC CGC AGT ATC ACC AAT GAC TCG GCT 1350 Met Lys Ser Asn His Met Arg Gly Arg Ser lie Thr Asn Asp Ser Ala 425 430 435

GTG CTG TCC CTC TTC CAG TCC ATC AAT AGC ACA CAC CCA CAG CTG CTC 1398 Val Leu Ser Leu Phe Gin Ser lie Asn Ser Thr His Pro Gin Leu Leu 440 445 450

GAG CTG CTC AAC CGG CTG GAT GAG CGC AGG CTG TAC TAC GAG GGG CTT 1446 Glu Leu Leu Asn Arg Leu Asp Glu Arg Arg Leu Tyr Tyr Glu Gly Leu 455 460 465

CAG GAC AAG CTG GCA CAG ATA CGT GAT GCC GAG GGC CCT GAG TGC CCT 1494 Gin Asp Lys Leu Ala Gin lie Arg Asp Ala Glu Gly Pro Glu Cys Pro 470 475 480

GCA GTG AAG AGC ACA GGG AGA AGC TGC GCC GGG CAG CTG AGG AGG CGG 1542 Ala Val Lys Ser Thr Gly Arg Ser Cys Ala Gly Gin Leu Arg Arg Arg 485 490 495 500 AGC GGT CAA CGT CAG ATC CAG CTG GCA CAG AAG CTG GAG ATC ATG AGA 1590 Ser Gly Gin Arg Gin lie Gin Leu Ala Gin Lys Leu Glu lie Met Arg 505 510 515

CAA AAG AAG CAG GAG TAT CTG GAG GTG CAG AGA CAG CTA GCT ATC CAG 1638 Gin Lys Lys Gin Glu Tyr Leu Glu Val Gin Arg Gin Leu Ala lie Gin 520 525 530

CGT CTG CAG GAA CAG GAG AAG GAA CGG CAG ATG CGC CTG GAG CAA CAG 1686 Arg Leu Gin Glu Gin Glu Lys Glu Arg Gin Met Arg Leu Glu Gin Gin 535 540 545

AAG CAG ACT GTC CAG ATG CGT GCC CAG ATG CCT GCC TTC CCC TTG CCT 1734 Lys Gin Thr Val Gin Met Arg Ala Gin Met Pro Ala Phe Pro Leu Pro 550 555 560

TAT GCC CAG CTC CAG GCT ATG CCA CAG CTG GGG GTG TAC TCT ACC AGC 1782 Tyr Ala Gin Leu Gin Ala Met Pro Gin Leu Gly Val Tyr Ser Thr Ser 565 570 575 580 CCT CAG GCC CAA CCA GCT TTC CTG GCA CCT TTA GCC CAG CAG GTA GTC 1830 Pro Gin Ala Gin Pro Ala Phe Leu Ala Pro Leu Ala Gin Gin Val Val 585 590 595

AGA GGG CTC TCC GAT GCA TGG TGT GTA TAT GAG CCA GCC AGC CCA GCA 1878 Arg Gly Leu Ser Asp Ala Trp Cys Val Tyr Glu Pro Ala Ser Pro Ala 600 605 610 CTG GCC CCT ACC CCA GCA TGC CTG GCA CCA CAG CAG ATC CCA GCA TGG 1926 Leu Ala Pro Thr Pro Ala Cys Leu Ala Pro Gin Gin lie Pro Ala Trp 615 620 625 TCA GCG ACT ACA TGT ACC CAG CAG GTG CCC TGG GGC ACA GGC AGC CCT 1974 Ser Ala Thr Thr Cys Thr Gin Gin Val Pro Trp Gly Thr Gly Ser Pro 630 635 640

CAG GCC AGG CCG GGC CAC CAC CAA CCC TGC TAC TCC TCG TAC CAG CCT 2022 Gin Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser Ser Tyr Gin Pro 645 650 655 660

ACT CCA ACC CCA GGC TAC CAG AAT GTG GCT TCT CAG GCC CCA CAG AGC 2070 Thr Pro Thr Pro Gly Tyr Gin Asn Val Ala Ser Gin Ala Pro Gin Ser 665 670 675

CTC CCA GCC ATC TCC CAG CCT CCA CAG ACC AGC AAC ATT GGC TAC ATG 2118 Leu Pro Ala lie Ser Gin Pro Pro Gin Thr Ser Asn lie Gly Tyr Met 680 685 690

GGG AGC CAG CCA ATG TCC ATG GGC TAC CAG CCA TAC AAC ATG CAG AAT 2166 Gly Ser Gin Pro Met Ser Met Gly Tyr Gin Pro Tyr Asn Met Gin Asn 695 700 705 CTC ATG ACC ACC CTT CCA GGC CAG GAT GCG TCT CTG CCA GCC CAG CAC 2214 Leu Met Thr Thr Leu Pro Gly Gin Asp Ala Ser Leu Pro Ala Gin His 710 715 720

CCC TAC ATC GCA GGC AGC AGC CCA TGT ACC AGC AGA TGG CAC CCA GCA 2262 Pro Tyr lie Ala Gly Ser Ser Pro Cys Thr Ser Arg Trp His Pro Ala

725 730 735 740

CTG GCC CTC CCC CAG CAG CAG CCC CCT TGG CCC AAC CGC CAC CTA CAC 2310 Leu Ala Leu Pro Gin Gin Gin Pro Pro Trp Pro Asn Arg His Leu His 745 750 755

AGG GAC CGC CAG CAC AGG GCA ATG AGA CCC AGC TCA TCT CCT TCG ACT 2358 Arg Asp Arg Gin His Arg Ala Met Arg Pro Ser Ser Ser Pro Ser Thr 760 765 770

GAC CTT GAG TCT GGC GCT CAC CAT CCA GAG TAC ACT ACA GTT CTC CAG 2406 Asp Leu Glu Ser Gly Ala His His Pro Glu Tyr Thr Thr Val Leu Gin 775 780 785 AAA CCA CTT ATA TGT CTA ACT AGC CAT TTC CTC CCA TTA CTG CCC TGT 2454

Lys Pro Leu lie Cys Leu Thr Ser His Phe Leu Pro Leu Leu Pro Cys 790 795 800

AGT GTC CCT TCT GTG AGC AAG GGG TGG GCC TTC ACC CTT GGC CCT CCT 2502 Ser Val Pro Ser Val Ser Lys Gly Trp Ala Phe Thr Leu Gly Pro Pro 805 810 815 820

CCC TGT CCT CAG TGG TCT GGC TCC TGT CAC TGG TTC CCT GCT TTG GTC 2550 Pro Cys Pro Gin Trp Ser Gly Ser Cys His Trp Phe Pro Ala Leu Val 825 830 835

CTG ATG CAG TCC GAC CTT CCC GGG ACT GGA CTC TGC ATG ACA AGT AGA 2598 Leu Met Gin Ser Asp Leu Pro Gly Thr Gly Leu Cys Met Thr Ser Arg 840 845 850

CCT TTT CTG GAG AAT GCC CAG CTG TGT CGG GGC CAT GCC AGA GGT GAC 2646 Pro Phe Leu Glu Asn Ala Gin Leu Cys Arg Gly His Ala Arg Gly Asp 855 860 865 TGC ATG TGG GGA TGG TTA GCC TGG CCC AGA GTC CGA TGT GAA CTG TGT 2694 Cys Met Trp Gly Trp Leu Ala Trp Pro Arg Val Arg Cys Glu Leu Cys 870 875 880 GGT GTC CAG CAT GGC CCT GGT ACC CAG AAG AAC GAT GTG ACC CAT GCA 2742 Gly Val Gin His Gly Pro Gly Thr Gin Lys Asn Asp Val Thr His Ala 885 890 895 900 CAG CAA GGG TGG AAG TTT CAG GCA TCT CTG TCT CCC CTA CTC CTT GGA 2790 Gin Gin Gly Trp Lys Phe Gin Ala Ser Leu Ser Pro Leu Leu Leu Gly 905 910 915

TGT CAT CTC TCC AGT GCA GAA CAG TGA 2817 Cys His Leu Ser Ser Ala Glu Gin 920

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 924 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : Met Gly Arg Gly Ser Gly Thr Phe Glu Arg Leu Leu Asp Lys Ala Thr 1 5 10 15

Ser Gin Leu Leu Leu Glu Thr Asp Trp Glu Ser lie Leu Gin lie Cys 20 25 30

Asp Leu lie Arg Gin Gly Asp Thr Gin Ala Lys Tyr Ala Val Asn Ser 35 40 45 lie Lys Lys Lys Val Asn Asp Lys Asn Pro His Val Ala Leu Tyr Ala 50 55 60

Leu Glu Val Met Glu Ser Val Val Lys Asn Cys Gly Gin Thr Val His 65 70 75 80 Asp Glu Val Ala Asn Lys Gin Thr Met Glu Glu Leu Lys Glu Leu Leu

85 90 95

Lys Arg Gin Val Glu Val Asn Val Arg Asn Lys lie Leu Tyr Leu lie 100 105 110

Gin Ala Trp Ala His Ala Phe Arg Asn Glu Pro Lys Tyr Lys Val Val 115 120 125

Gin Asp Thr Tyr Gin lie Met Lys Val Glu Gly His Val Phe Pro Glu 130 135 140

Phe Lys Glu Ser Asp Ala Met Phe Ala Ala Glu Arg Ala Pro Asp Trp 145 150 155 160 Val Asp Ala Glu Glu Cys His Arg Cys Arg Val Gin Phe Gly Val Val

165 170 175

Thr Arg Lys His His Cys Arg Ala Cys Gly Gin lie Phe Cys Gly Lys 180 185 190

Cys Ser Ser Lys Tyr Ser Thr He Pro Lys Phe Gly He Glu Lys Glu 195 200 205

Val Arg Val Cys Glu Pro Cys Tyr Glu Gin Leu Asn Lys Lys Ala Glu 210 215 220

Gly Lys Ala Ala Ser Thr Thr Glu Leu Pro Pro Glu Tyr Leu Thr Ser 225 230 235 240

Pro Leu Ser Gin Gin Ser Gin Leu Pro Pro Lys Arg Asp Glu Thr Cys 245 250 255

Ala Ala Gly Arg Gly Gly Ala Thr Ala Gly Ser Gly His Ser Gin Ser 260 265 270

Glu Ala Glu Glu Lys Glu Arg Met Arg Gin Lys Ser Thr Tyr Thr Ala 275 280 285

His Pro Lys Ser Glu Pro Ala Pro Leu Ala Ser Ser Ala Pro Pro Ala 290 295 300 Gly Ser Leu Tyr Ser Ser Pro Val Asn Ser Ser Ala Pro Leu Ala Glu 305 310 315 320

Asp He Asp Pro Glu Leu Ala Arg Tyr Leu Asn Arg Asn Tyr Trp Glu 325 330 335

Lys Lys Gin Glu Glu Ala Arg Lys Ser Pro Thr Pro Ser Ala Pro Val 340 345 350

Pro Leu Thr Glu Pro Ala Ala Gin Pro Gly Glu Gly His Thr Ala Pro 355 360 365

Asn Ser Met Val Glu Ala Pro Leu Pro Glu Thr Asp Ser Gin Pro He 370 375 380 Thr Ser Cys Ser Gly Pro Phe Ser Glu Tyr Gin Asn Gly Glu Ser Glu 385 390 395 400

Glu Ser His Glu Gin Phe Leu Lys Ala Leu Gin Asn Ala Val Ser Thr 405 410 415

Phe Val Asn Arg Met Lys Ser Asn His Met Arg Gly Arg Ser He Thr 420 425 430

Asn Asp Ser Ala Val Leu Ser Leu Phe Gin Ser He Asn Ser Thr His 435 440 445

Pro Gin Leu Leu Glu Leu Leu Asn Arg Leu Asp Glu Arg Arg Leu Tyr 450 455 460 Tyr Glu Gly Leu Gin Asp Lys Leu Ala Gin He Arg Asp Ala Glu Gly 465 470 475 480

Pro Glu Cys Pro Ala Val Lys Ser Thr Gly Arg Ser Cys Ala Gly Gin 485 490 495

Leu Arg Arg Arg Ser Gly Gin Arg Gin He Gin Leu Ala Gin Lys Leu 500 505 510

Glu He Met Arg Gin Lys Lys Gin Glu Tyr Leu Glu Val Gin Arg Gin 515 520 525

Leu Ala He Gin Arg Leu Gin Glu Gin Glu Lys Glu Arg Gin Met Arg 530 535 540 Leu Glu Gin Gin Lys Gin Thr Val Gin Met Arg Ala Gin Met Pro Ala 545 550 555 560

Phe Pro Leu Pro Tyr Ala Gin Leu Gin Ala Met Pro Gin Leu Gly Val 565 570 575

Tyr Ser Thr Ser Pro Gin Ala Gin Pro Ala Phe Leu Ala Pro Leu Ala 580 585 590 Gin Gin Val Val Arg Gly Leu Ser Asp Ala Trp Cys Val Tyr Glu Pro 595 600 605

Ala Ser Pro Ala Leu Ala Pro Thr Pro Ala Cys Leu Ala Pro Gin Gin 610 615 620

He Pro Ala Trp Ser Ala Thr Thr Cys Thr Gin Gin Val Pro Trp Gly 625 630 635 640 Thr Gly Ser Pro Gin Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser

645 650 655

Ser Tyr Gin Pro Thr Pro Thr Pro Gly Tyr Gin Asn Val Ala Ser Gin

660 665 670

Ala Pro Gin Ser Leu Pro Ala He Ser Gin Pro Pro Gin Thr Ser Asn

675 680 685

He Gly Tyr Met Gly Ser Gin Pro Met Ser Met Gly Tyr Gin Pro Tyr 690 695 700

Asn Met Gin Asn Leu Met Thr Thr Leu Pro Gly Gin Asp Ala Ser Leu 705 710 715 720 Pro Ala Gin His Pro Tyr He Ala Gly Ser Ser Pro Cys Thr Ser Arg

725 730 735

Trp His Pro Ala Leu Ala Leu Pro Gin Gin Gin Pro Pro Trp Pro Asn 740 745 750

Arg His Leu His Arg Asp Arg Gin His Arg Ala Met Arg Pro Ser Ser 755 760 765

Ser Pro Ser Thr Asp Leu Glu Ser Gly Ala His His Pro Glu Tyr Thr 770 775 780

Thr Val Leu Gin Lys Pro Leu He Cys Leu Thr Ser His Phe Leu Pro 785 790 795 800 Leu Leu Pro Cys Ser Val Pro Ser Val Ser Lys Gly Trp Ala Phe Thr

805 810 815

Leu Gly Pro Pro Pro Cys Pro Gin Trp Ser Gly Ser Cys His Trp Phe 820 825 830

Pro Ala Leu Val Leu Met Gin Ser Asp Leu Pro Gly Thr Gly Leu Cys 835 840 845

Met Thr Ser Arg Pro Phe Leu Glu Asn Ala Gin Leu Cys Arg Gly His 850 855 860

Ala Arg Gly Asp Cys Met Trp Gly Trp Leu Ala Trp Pro Arg Val Arg 865 870 875 880 Cys Glu Leu Cys Gly Val Gin His Gly Pro Gly Thr Gin Lys Asn Asp

885 890 895

Val Thr His Ala Gin Gin Gly Trp Lys Phe Gin Ala Ser Leu Ser Pro 900 905 910

Leu Leu Leu Gly Cys His Leu Ser Ser Ala Glu Gin 915 920

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2328 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Human Hrs-2 cDNA (partial) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2325

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GCC ATG TTT GCT GCT GAA AGA GCC CCT GAC TGG GTG GAC GCT GAG GAA 48 Ala Met Phe Ala Ala Glu Arg Ala Pro Asp Trp Val Asp Ala Glu Glu 1 5 10 15

TGC CAC CGC TGC AGG GTG CAG TTC GGG GTG ATG ACC CGT AAG CAC CAC 96 Cys His Arg Cys Arg Val Gin Phe Gly Val Met Thr Arg Lys His His 20 25 30 TGC CGG GAG TGT GGG CAG ATC ATC TGT GGA AAG TGT TCT TCC AAG TAC 144 Cys Arg Glu Cys Gly Gin He He Cys Gly Lys Cys Ser Ser Lys Tyr 35 40 45

TCC ACC ATC CCC AAG TTT GGC ATT GAG AAG GAG GTG CGC GTG TGT GAG 192 Ser Thr He Pro Lys Phe Gly He Glu Lys Glu Val Arg Val Cys Glu 50 55 60

CCC TGC TAC GAG CAG CTG AAC AGG AAA GCA GAG GGA AAG GCT GCC ACT 240 Pro Cys Tyr Glu Gin Leu Asn Arg Lys Ala Glu Gly Lys Ala Ala Thr 65 70 75 80

ACC ACT GAG CTG CCC CCC GAG TAC CTG ACC AGC CCC CTG TCT CAG CAG 288 Thr Thr Glu Leu Pro Pro Glu Tyr Leu Thr Ser Pro Leu Ser Gin Gin 85 90 95

TCC CAG CTG CCC CCA AAG CGG GAC GAG ACA TGC GCT GCA GGA GGA GGA 336 Ser Gin Leu Pro Pro Lys Arg Asp Glu Thr Cys Ala Ala Gly Gly Gly 100 105 110 GGA GCT GCA GCT GGC TCT GGC CAT TCA CAG TCA GAG GCG GAG GAG AAG 384 Gly Ala Ala Ala Gly Ser Gly His Ser Gin Ser Glu Ala Glu Glu Lys 115 120 125

GAG AGG ATG AGA CAG AAG TCC ACG TAC ACT GCG CAT CCC AAG GCG GCG 432 Glu Arg Met Arg Gin Lys Ser Thr Tyr Thr Ala His Pro Lys Ala Ala 130 135 140

ACT GCG CCC TTG GCT TCC TCT GCA CCC CCC GCC AGC AGC CTG TAC TCT 480 Thr Ala Pro Leu Ala Ser Ser Ala Pro Pro Ala Ser Ser Leu Tyr Ser 145 150 155 160

TCA CCT GTG AAC TCG TCA GCA CCT CTG GCT GAG GAC ATC GAC CCT GAG 528 Ser Pro Val Asn Ser Ser Ala Pro Leu Ala Glu Asp He Asp Pro Glu 165 170 175

CTC GCA AGA TAC CTC AAC CGG AAC TAC TGG GAG AAG AAG CAG GAG GAG 576 Leu Ala Arg Tyr Leu Asn Arg Asn Tyr Trp Glu Lys Lys Gin Glu Glu 180 185 190

GCT CGC AAG AGC CCC ACA CCA TCT GCA CCT GTG CCC CTG ACC GAG CCA 624 Ala Arg Lys Ser Pro Thr Pro Ser Ala Pro Val Pro Leu Thr Glu Pro 195 200 205

GCT GCC CAG CCC GGA GAA GGA CAT ACA GCC CCC AAC AGC ATG GTA GAG 672 Ala Ala Gin Pro Gly Glu Gly His Thr Ala Pro Asn Ser Met Val Glu 210 215 220

GCC CCT CTT CCA GAG ACA GAC TCT CAG CCC ATA ACT TCC TGC AGT GGC 720 Ala Pro Leu Pro Glu Thr Asp Ser Gin Pro He Thr Ser Cys Ser Gly 225 230 235 240 CCC TTT AGT GAG TAC CAG AAC GGG GAG TCG GAG GAG AGC CAC GAG CAG 768 Pro Phe Ser Glu Tyr Gin Asn Gly Glu Ser Glu Glu Ser His Glu Gin 245 250 255

TTC CTC AAG GCC CTG CAG AAT GCA GTC AGC ACT TTT GTC AAC CGC ATG 816 Phe Leu Lys Ala Leu Gin Asn Ala Val Ser Thr Phe Val Asn Arg Met 260 265 270

AAG AGC AAC CAC ATG AGG GGC CGC AGT ATC ACC AAT GAC TCG GCT GTG 864 Lys Ser Asn His Met Arg Gly Arg Ser He Thr Asn Asp Ser Ala Val 275 280 285

CTG TCC CTC TTC CAG TCC ATC AAT AGC ACA CAC CCA CAG CTG CTC GAG 912 Leu Ser Leu Phe Gin Ser He Asn Ser Thr His Pro Gin Leu Leu Glu 290 295 300

CTG CTC AAC CGG CTG GAT GAG CGC AGG CTG TAC TAC GAG GGG CTT CAG 960 Leu Leu Asn Arg Leu Asp Glu Arg Arg Leu Tyr Tyr Glu Gly Leu Gin 305 310 315 320 GAC AAG CTG GCA CAG ATA CGT GAT GCC GAG GGC CCT GAG TGC CCT GCA 1008 Asp Lys Leu Ala Gin He Arg Asp Ala Glu Gly Pro Glu Cys Pro Ala 325 330 335

GTG AAG AGC ACA GGG AGA AGC TGC GCC GGG CAG CTG AGG AGG CGG AGC 1056 Val Lys Ser Thr Gly Arg Ser Cys Ala Gly Gin Leu Arg Arg Arg Ser 340 345 350

GGT CAA CGT CAG ATC CAG CTG GCA CAG AAG CTG GAG ATC ATG AGA CAA 1104 Gly Gin Arg Gin He Gin Leu Ala Gin Lys Leu Glu He Met Arg Gin 355 360 365

AAG AAG CAG GAG TAT CTG GAG GTG CAG AGA CAG CTA GCT ATC CAG CGT 1152 Lys Lys Gin Glu Tyr Leu Glu Val Gin Arg Gin Leu Ala He Gin Arg 370 375 380

CTG CAG GAA CAG GAG AAG GAA CGG CAG ATG CGC CTG GAG CAA CAG AAG 1200 Leu Gin Glu Gin Glu Lys Glu Arg Gin Met Arg Leu Glu Gin Gin Lys 385 390 395 400 CAG ACT GTC CAG ATG CGT GCC CAG ATG CCT GCC TTC CCC TTG CCT TAT 1248 Gin Thr Val Gin Met Arg Ala Gin Met Pro Ala Phe Pro Leu Pro Tyr 405 410 415

GCC CAG CTC CAG GCT ATG CCA CAG CTG GGG GTG TAC TCT ACC AGC CCT 1296 Ala Gin Leu Gin Ala Met Pro Gin Leu Gly Val Tyr Ser Thr Ser Pro 420 425 430

CAG GCC CAA CCA GCT TTC CTG GCA CCT TTA GCC CAG CAG GTA GTC AGA 1344 Gin Ala Gin Pro Ala Phe Leu Ala Pro Leu Ala Gin Gin Val Val Arg 435 440 445

GGG CTC TCC GAT GCA TGG TGT GTA TAT GAG CCA GCC AGC CCA GCA CTG 1392 Gly Leu Ser Asp Ala Trp Cys Val Tyr Glu Pro Ala Ser Pro Ala Leu 450 455 460

GCC CCT ACC CCA GCA TGC CTG GCA CCA CAG CAG ATC CCA GCA TGG TCA 1440 Ala Pro Thr Pro Ala Cys Leu Ala Pro Gin Gin He Pro Ala Trp Ser 465 470 475 480

GCG ACT ACA TGT ACC CAG CAG GTG CCC TGG GGC ACA GGC AGC CCT CAG 1488 Ala Thr Thr Cys Thr Gin Gin Val Pro Trp Gly Thr Gly Ser Pro Gin 485 490 495

GCC AGG CCG GGC CAC CAC CAA CCC TGC TAC TCC TCG TAC CAG CCT ACT 1536 Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser Ser Tyr Gin Pro Thr 500 505 510

CCA ACC CCA GGC TAC CAG AAT GTG GCT TCT CAG GCC CCA CAG AGC CTC 1584 Pro Thr Pro Gly Tyr Gin Asn Val Ala Ser Gin Ala Pro Gin Ser Leu 515 520 525 CCA GCC ATC TCC CAG CCT CCA CAG ACC AGC AAC ATT GGC TAC ATG GGG 1632 Pro Ala He Ser Gin Pro Pro Gin Thr Ser Asn He Gly Tyr Met Gly 530 535 540

AGC CAG CCA ATG TCC ATG GGC TAC CAG CCA TAC AAC ATG CAG AAT CTC 1680 Ser Gin Pro Met Ser Met Gly Tyr Gin Pro Tyr Asn Met Gin Asn Leu 545 550 555 560

ATG ACC ACC CTT CCA GGC CAG GAT GCG TCT CTG CCA GCC CAG CAC CCC 1728 Met Thr Thr Leu Pro Gly Gin Asp Ala Ser Leu Pro Ala Gin His Pro 565 570 575

TAC ATC GCA GGC AGC AGC CCA TGT ACC AGC AGA TGG CAC CCA GCA CTG 1776 Tyr He Ala Gly Ser Ser Pro Cys Thr Ser Arg Trp His Pro Ala Leu 580 585 590

GCC CTC CCC CAG CAG CAG CCC CCT TGG CCC AAC CGC CAC CTA CAC AGG 1824 Ala Leu Pro Gin Gin Gin Pro Pro Trp Pro Asn Arg His Leu His Arg 595 600 605 GAC CGC CAG CAC AGG GCA ATG AGG CCC AGC TCA TTT CAT TCG ACT GAC 1872 Asp Arg Gin His Arg Ala Met Arg Pro Ser Ser Phe His Ser Thr Asp 610 615 620

CTT GAG TCT GGC GCT CAC CAT CCA GAG TAC ACT ACA GTT CTC CAG AAA 1920 Leu Glu Ser Gly Ala His His Pro Glu Tyr Thr Thr Val Leu Gin Lys 625 630 635 640

CCA CTT ATA TGT CTA ACT AGC CAT TTC CTC CCA TTA CTG CCC TGT AGT 1968 Pro Leu He Cys Leu Thr Ser His Phe Leu Pro Leu Leu Pro Cys Ser 645 650 655

GTC CCT TCT GTG AGC AAG GGG TGG GCC TTC ACC CTT GGC CCT CCT CCC 2016 Val Pro Ser Val Ser Lys Gly Trp Ala Phe Thr Leu Gly Pro Pro Pro 660 665 670

TGT CCT CAG TGG TCT GGC TCC TGT CAC TGG TTC CCT GCT TTG GTC CTG 2064 Cys Pro Gin Trp Ser Gly Ser Cys His Trp Phe Pro Ala Leu Val Leu 675 680 685 ATG CAG TCC GAC CTT CCC GGG ACT GGA CTC TGC ATG ACA AGT AGA CCT 2112 Met Gin Ser Asp Leu Pro Gly Thr Gly Leu Cys Met Thr Ser Arg Pro 690 695 700

TTT CTG GAG AAT GCC CAG CTG TGT CGG GGC CAT GCC AGA GGT GAC TGC 2160 Phe Leu Glu Asn Ala Gin Leu Cys Arg Gly His Ala Arg Gly Asp Cys 705 710 715 720 ATG TGG GGA TGG TTA CTC TGG CCG CAC TGT GAG CTG GCT GTG GTG TCT 2208 Met Trp Gly Trp Leu Leu Trp Pro His Cys Glu Leu Ala Val Val Ser 725 730 735 GGG TGT CGC CTG GGG CTC CCT CTG CAG GGG CCT CTC TCG GCA GCC ACA 2256 Gly Cys Arg Leu Gly Leu Pro Leu Gin Gly Pro Leu Ser Ala Ala Thr 740 745 750

GCC AAG GGT GGA GGC TTC AGG TCT CCA GCT TCT CTC CTC TCA GCT GCC 2304 Ala Lys Gly Gly Gly Phe Arg Ser Pro Ala Ser Leu Leu Ser Ala Ala 755 760 765

ATC TCC AGT GCC CCA GAA TGG TAA 2328

He Ser Ser Ala Pro Glu Trp 770 775

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 775 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Ala Met Phe Ala Ala Glu Arg Ala Pro Asp Trp Val Asp Ala Glu Glu 1 5 10 15

Cys His Arg Cys Arg Val Gin Phe Gly Val Met Thr Arg Lys His His 20 25 30 Cys Arg Glu Cys Gly Gin He He Cys Gly Lys Cys Ser Ser Lys Tyr 35 40 45

Ser Thr He Pro Lys Phe Gly He Glu Lys Glu Val Arg Val Cys Glu 50 55 60

Pro Cys Tyr Glu Gin Leu Asn Arg Lys Ala Glu Gly Lys Ala Ala Thr 65 70 75 80

Thr Thr Glu Leu Pro Pro Glu Tyr Leu Thr Ser Pro Leu Ser Gin Gin 85 90 95

Ser Gin Leu Pro Pro Lys Arg Asp Glu Thr Cys Ala Ala Gly Gly Gly 100 105 110 Gly Ala Ala Ala Gly Ser Gly His Ser Gin Ser Glu Ala Glu Glu Lys 115 120 125

Glu Arg Met Arg Gin Lys Ser Thr Tyr Thr Ala His Pro Lys Ala Ala 130 135 140

Thr Ala Pro Leu Ala Ser Ser Ala Pro Pro Ala Ser Ser Leu Tyr Ser 145 150 155 160

Ser Pro Val Asn Ser Ser Ala Pro Leu Ala Glu Asp He Asp Pro Glu 165 170 175

Leu Ala Arg Tyr Leu Asn Arg Asn Tyr Trp Glu Lys Lys Gin Glu Glu 180 185 190 Ala Arg Lys Ser Pro Thr Pro Ser Ala Pro Val Pro Leu Thr Glu Pro 195 200 205 Ala Ala Gin Pro Gly Glu Gly His Thr Ala Pro Asn Ser Met Val Glu 210 215 220

Ala Pro Leu Pro Glu Thr Asp Ser Gin Pro He Thr Ser Cys Ser Gly 225 230 235 240

Pro Phe Ser Glu Tyr Gin Asn Gly Glu Ser Glu Glu Ser His Glu Gin 245 250 255 Phe Leu Lys Ala Leu Gin Asn Ala Val Ser Thr Phe Val Asn Arg Met 260 265 270

Lys Ser Asn His Met Arg Gly Arg Ser He Thr Asn Asp Ser Ala Val 275 280 285

Leu Ser Leu Phe Gin Ser He Asn Ser Thr His Pro Gin Leu Leu Glu 290 295 300

Leu Leu Asn Arg Leu Asp Glu Arg Arg Leu Tyr Tyr Glu Gly Leu Gin 305 310 315 320

Asp Lys Leu Ala Gin He Arg Asp Ala Glu Gly Pro Glu Cys Pro Ala 325 330 335 Val Lys Ser Thr Gly Arg Ser Cys Ala Gly Gin Leu Arg Arg Arg Ser 340 345 350

Gly Gin Arg Gin He Gin Leu Ala Gin Lys Leu Glu He Met Arg Gin 355 360 365

Lys Lys Gin Glu Tyr Leu Glu Val Gin Arg Gin Leu Ala He Gin Arg 370 375 380

Leu Gin Glu Gin Glu Lys Glu Arg Gin Met Arg Leu Glu Gin Gin Lys 385 390 395 400

Gin Thr Val Gin Met Arg Ala Gin Met Pro Ala Phe Pro Leu Pro Tyr 405 410 415 Ala Gin Leu Gin Ala Met Pro Gin Leu Gly Val Tyr Ser Thr Ser Pro 420 425 430

Gin Ala Gin Pro Ala Phe Leu Ala Pro Leu Ala Gin Gin Val Val Arg 435 440 445

Gly Leu Ser Asp Ala Trp Cys Val Tyr Glu Pro Ala Ser Pro Ala Leu 450 455 460

Ala Pro Thr Pro Ala Cys Leu Ala Pro Gin Gin He Pro Ala Trp Ser 465 470 475 480

Ala Thr Thr Cys Thr Gin Gin Val Pro Trp Gly Thr Gly Ser Pro Gin 485 490 495 Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser Ser Tyr Gin Pro Thr 500 505 510

Pro Thr Pro Gly Tyr Gin Asn Val Ala Ser Gin Ala Pro Gin Ser Leu 515 520 525

Pro Ala He Ser Gin Pro Pro Gin Thr Ser Asn He Gly Tyr Met Gly 530 535 540

Ser Gin Pro Met Ser Met Gly Tyr Gin Pro Tyr Asn Met Gin Asn Leu 545 550 555 560

Met Thr Thr Leu Pro Gly Gin Asp Ala Ser Leu Pro Ala Gin His Pro 565 570 575

Tyr He Ala Gly Ser Ser Pro Cys Thr Ser Arg Trp His Pro Ala Leu 580 585 590

Ala Leu Pro Gin Gin Gin Pro Pro Trp Pro Asn Arg His Leu His Arg 595 600 605

Asp Arg Gin His Arg Ala Met Arg Pro Ser Ser Phe His Ser Thr Asp 610 615 620

Leu Glu Ser Gly Ala His His Pro Glu Tyr Thr Thr Val Leu Gin Lys 625 630 635 640 Pro Leu He Cys Leu Thr Ser His Phe Leu Pro Leu Leu Pro Cys Ser

645 650 655

Val Pro Ser Val Ser Lys Gly Trp Ala Phe Thr Leu Gly Pro Pro Pro 660 665 670

Cys Pro Gin Trp Ser Gly Ser Cys His Trp Phe Pro Ala Leu Val Leu 675 680 685

Met Gin Ser Asp Leu Pro Gly Thr Gly Leu Cys Met Thr Ser Arg Pro 690 695 700

Phe Leu Glu Asn Ala Gin Leu Cys Arg Gly His Ala Arg Gly Asp Cys 705 710 715 720 Met Trp Gly Trp Leu Leu Trp Pro His Cys Glu Leu Ala Val Val Ser

725 730 735

Gly Cys Arg Leu Gly Leu Pro Leu Gin Gly Pro Leu Ser Ala Ala Thr 740 745 750

Ala Lys Gly Gly Gly Phe Arg Ser Pro Ala Ser Leu Leu Ser Ala Ala 755 760 765

He Ser Ser Ala Pro Glu Trp 770 775

(2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: putative nucleotide binding site GXXXXGK

( ix) FEATURE :

(A) NAME/KEY: Modified-site

(B) LOCATION: 2..5

(D) OTHER INFORMATION: /note= "where Xaa is any amino acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gly Xaa Xaa Xaa Xaa Gly Lys 1 5

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: nucleotide binding site RDET

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : Arg Asp Glu Thr

1

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: nucleotide binding site DXXG

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2..3

(D) OTHER INFORMATION: /note= "where Xaa is any amino acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Asp Xaa Xaa Gly 1

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL : NO ( iv) ANTI - SENSE : NO (vi ) ORIGINAL SOURCE :

( C) INDIVIDUAL ISOLATE : nucleotide binding site TQ XD

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 4..4

(D) OTHER INFORMATION: /note= "where Xaa is any amino acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Thr Gin Lys Xaa Asp 1 5

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2040 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Mouse SNAP-25 (GenBank M22012)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 164..784

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CCCGAGGTTT GGAGCTGTCT TTCCTTCCCT CCCTACCCGG CGGCTCCTCC ACTCTTGCTA 60 CCTGCAGGGA TCAGCGGACA GCATCCTCTG AAGAAGACAA GGTTCCTTAA CTAAGCACCA 120

CTGACTTGCT GGCCCCGGCG CCCAGCAACC CCCCACCACT ACC ATG GCC GAG GAC 175

Met Ala Glu Asp 1

GCA GAC ATG CGT AAT GAA CTG GAG GAG ATG CAG AGG AGG GCT GAC CAG 223 Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gin Arg Arg Ala Asp Gin 5 10 15 20 CTG GCT GAT GAG TCC CTG GAA AGC ACC CGT CGC ATG CTG CAG CTG GTC 271 Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met Leu Gin Leu Val 25 30 35

GAA GAG AGT AAA GAT GCT GGC ATC AGG ACT TTG GTT ATG TTG GAT GAG 319 Glu Glu Ser Lys Asp Ala Gly He Arg Thr Leu Val Met Leu Asp Glu

40 45 50

CAA GGC GAA CAA CTG GAA CGC ATT GAG GAA GGG ATG GAC CAA ATC AAT 367 Gin Gly Glu Gin Leu Glu Arg He Glu Glu Gly Met Asp Gin He Asn 55 60 65

AAG GAT ATG AAA GAA GCA GAA AAG AAT TTG ACG GAC CTA GGA AAA TTC 415 Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp Leu Gly Lys Phe 70 75 80

TGC GGG CTT TGT GTG TGT CCC TGT AAC AAG CTT AAA TCC AGT GAT GCT 463 Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys Ser Ser Asp Ala 85 90 95 100

TAC AAA AAA GCC TGG GGC AAT AAT CAG GAT GGA GTA GTG GCC AGC CAG 511 Tyr Lys Lys Ala Trp Gly Asn Asn Gin Asp Gly Val Val Ala Ser Gin 105 110 115

CCT GCC CGT GTG GTG GAT GAA CGG GAG CAG ATG GCC ATC AGT GGT GGC 559 Pro Ala Arg Val Val Asp Glu Arg Glu Gin Met Ala He Ser Gly Gly 120 125 130

TTC ATC CGC AGG GTA ACA AAC GAT GCC CGG GAA AAT GAA ATG GAT GAA 607 Phe He Arg Arg Val Thr Asn Asp Ala Arg Glu Asn Glu Met Asp Glu 135 140 145 AAC CTA GAG CAG GTG AGC GGC ATC ATC GGA AAC CTC CGT CAT ATG GCC 655 Asn Leu Glu Gin Val Ser Gly He He Gly Asn Leu Arg His Met Ala 150 155 160

CTA GAC ATG GGC AAT GAG ATT GAC ACC CAG AAT CGC CAG ATT GAC AGG 703 Leu Asp Met Gly Asn Glu He Asp Thr Gin Asn Arg Gin He Asp Arg 165 170 175 180

ATC ATG GAG AAG GCT GAC TCC AAC AAA ACC AGA ATT GAT GAA GCC AAC 751 He Met Glu Lys Ala Asp Ser Asn Lys Thr Arg He Asp Glu Ala Asn 185 190 195

CAA CGT GCA ACA AAG ATG CTG GGA AGT GGT TAAATCTGCC GTTCTGCTGT 801 Gin Arg Ala Thr Lys Met Leu Gly Ser Gly 200 205

GCTGTCCTCC AATGTTGTTG GACAAGAGAG AAGAGAGCTC CTTCATGCTT CTCTCATGGT 861

ATTACCTAGT AAGACTTACA CACACACACA CACACACACA CACACACACA CACACACACA 921 CACACACACA GAGTAGTCAC CCCCATTGTA AATGTCTGTG TGGTTTGTCA GCTTCCCAAT 981

GATACCATGT GTCTTTTGTT TTCTCCGGCT CTCTTTCTTT GCCAAAGGTT GTACATAGTG 1041

GTCATCTGGT GACTCTATTT CCTGACTTAA GAGTTCTTGG GTCTCTCTCT TTCTTTTCTC 1101

AGTGGCGTTT GCTGAATGAC AACAATTTAG GAATGCTCAA TGTACTGTTG ATTTTTCTCA 1161

ATACACAGTA TTGTTCTTGT AAAACTGTGA CTTACCACAG AGCTACTACC ACAGTCCTTT 1221 CTTAGGGTGT CAGGCTCTGA ATCTCTCCAA ATGTGCTCTC TTTGGTTCCT CAGTGCTATT 1281

CTTTGTCTTT ATGATTTCAT AATTAGACAA TGTGAAATTA CATAACAGGC ATTGCACTAA 1341

AAGTGATGTG ATTTATGCAT TTATGCATGA GAACTAAATA GACTTTTAGA TCCTACTTAA 1401

ACAAAAACTT CCATGACAGT AGCATACTGA CAAGAAAACA CACACAACAG CAACAATAAC 1461

AAAGCAACAA CTACGCATGC TCAGCATTGG GACACTGTCA AGATTAAGTC ATACCAGCAA 1521 AACCTGCAGC TGTGTCACCT TCTTCTGTCA ACATACAGAC TGATCATAAT GATCCCTTCT 1581

TTACACACAC ACACACACAC ACACACACAC ACACACACAC AAATGGAATT TAACCAACTT 1641

CCCAGAATTG ATGAAGCAAA TATATGTTTG GCTGAAACTA TTGTAAATGG GTGTAATATA 1701

GGGTTTGTCG AATGCTTTTG AAAGCTCTGT TTTCCAGACA ATACTCTTGT GTGGAAAACG 1761 TGAAGATCTT CTAAGTCTGG CTCTTGTGAT CACCAAACCC TGGTGCATCA GTACAACACT 1821

TTGCGCTAAT CTAGAGCTAT GCACAACCAA ATTGCTGAGA TGTTTAGTAG CTGATAAAGA 1881 AACCTTTAAA AAATTATATA AATGAATGAA ATATAGATAA ACTGTGAGAT AAATATCATT 1941

ACAGCATGTA TATTAAATCC CTCCTGTCTC CTCTGTTGGT TTGTGAAGTG ATTTGACATT 2001

TTGTAGCTAG TTTAAAATTA TTAAAAATTA TAGATGTTA 2040

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 206 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :

Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gin Arg 1 5 10 15

Arg Ala Asp Gin Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30

Leu Gin Leu Val Glu Glu Ser Lys Asp Ala Gly He Arg Thr Leu Val 35 40 45

Met Leu Asp Glu Gin Gly Glu Gin Leu Glu Arg He Glu Glu Gly Met 50 55 60 Asp Gin He Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp 65 70 75 80

Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90 95

Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gin Asp Gly Val 100 105 110

Val Ala Ser Gin Pro Ala Arg Val Val Asp Glu Arg Glu Gin Met Ala 115 120 125

He Ser Gly Gly Phe He Arg Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140 Glu Met Asp Glu Asn Leu Glu Gin Val Ser Gly He He Gly Asn Leu 145 150 155 160

Arg His Met Ala Leu Asp Met Gly Asn Glu He Asp Thr Gin Asn Arg 165 170 175

Gin He Asp Arg He Met Glu Lys Ala Asp Ser Asn Lys Thr Arg He 180 185 190

Asp Glu Ala Asn Gin Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200 205

Claims

IT IS CLAIMED:

I. A substantially purified Hrs-2 polypeptide.

2. The polypeptide of claim 1, wherein the polypeptide is encoded by a polynucleotide sequence derived from the genome of a rat.

3. The polypeptide of claim 2, wherein the polypeptide contains a region of at least 10 consecutive amino acids corresponding to a region contained in SEQ ID NO:4.

4. The polypeptide of claim 3, wherein the polypeptide contains the sequence represented as SEQ ID NO:4.

5. The polypeptide of claim 1, wherein the polypeptide is encoded by a polynucleotide sequence derived from the genome of a human.

6. The polypeptide of claim 5, wherein the polypeptide contains a region of at least 10 consecutive amino acids corresponding to a region contained in SEQ ID NO:6.

7. The polypeptide of claim 6, wherein the polypeptide contains the sequence represented as SEQ ID NO:6.

8. A substantially purified Hrs-2 polynucleotide.

9. The polynucleotide of claim 8, wherein the sequence of the polynucleotide is derived from the genome of a rat.

10. The polynucleotide of claim 9, wherein the polynucleotide contains a region of at least 20 consecutive nucleotides corresponding to a region contained in SEQ ID NO:3.

II. The polynucleotide of claim 10, wherein the polynucleotide contains the sequence represented as SEQ ID NO:3.

12. The polynucleotide of claim 8, wherein the sequence of the polynucleotide is derived from the genome of a human.

13. The polynucleotide of claim 12, wherein the polynucleotide contains a region of at least 20 consecutive nucleotides corresponding to a region contained in SEQ ID NO:5.

14. The polynucleotide of claim 13, wherein the polynucleotide contains the sequence represented as SEQ ID NO:5.

15. A method of identifying a compound capable of modulating calcium-regulated secretion of secretory vesicles, comprising contacting a SNAP-25 polypeptide with an Hrs-2 polypeptide, in the presence and absence of a test compound, measuring the effect of the test compound on the extent of binding between the SNAP-25 and Hrs-2 polypeptides, and identifying said compound as effective if its measured effect on the extent of binding is above a threshold level.

16. The method of claim 15, wherein said SNAP-25 polypeptide has the sequence represented as SEQ ID NO: 11.

17. The method of claim 15, wherein said Hrs-2 polypeptide has the sequence represented as SEQ ID NO:4.

18. The method of claim 15, wherein said Hrs-2 polypeptide has the sequence represented as SEQ ID NO:6.

19. The method of claim 15, wherein said Hrs-2 polypeptide has the sequence represented as SEQ ID NO: 8.

20. The method of claim 15, wherein said test compound inhibits the binding between the SNAP-25 and Hrs-2 polypeptides.

21. The method of claim 15, wherein said test compound potentiates the binding between the SNAP-25 and Hrs-2 polypeptides.

22. The method of claim 15, wherein said test compound is one of a plurality of small molecules in a small molecule combinatorial library.

23. The method of claim 15, wherein said test compound is a peptide.

24. The method of claim 15, wherein said secretory vesicles are neurotransmitter- containing synaptic vesicles.