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The Journal of Neuroscience, 2000, 20:RC59:1-5

RAPID COMMUNICATION
Copurification of Brain G-Protein 5 with RGS6 and RGS7

Jian-Hua Zhang and William F. Simonds

Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A structurally divergent G-protein  subunit expressed in brain and retina, G₅, exhibits functional specialization in its protein-protein interactions in vitro. In retina, G5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of G₅ in brain are unknown. To identify tightly bound proteins associated with G₅ in the brain, it was immunoaffinity-purified from a nonionic detergent extract of washed mouse brain membranes using an antibody directed against its N terminus. Elution with cognate peptide revealed a broad band of 55 kDa that coeluted with G₅ on SDS-PAGE. The copurifying 55 kDa band was identified as a ~1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. G₅ and RGS7 could be reciprocally coimmunoprecipitated from unfractionated brain membrane extracts confirming the tight association of native proteins. In contrast, immunoblotting of the peptide eluate revealed no copurifying Gq/11, Gi1/2, G2, G3, or G7. These findings implicate RGS6 and RGS7 in the function of G₅ in the brain and suggest that a large fraction of membrane-targeted G₅ has no associated G subunit and therefore functions outside the canonical framework of G interactions.

Key words: signal transduction; G-proteins; regulators of G-protein signaling; Igs; affinity purification; mass spectroscopy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mediate signals generated after activation of seven transmembrane-spanning receptors in eukaryotic cells. The G complex of G-proteins may regulate effectors independently of the G subunits, so that after activation, G-proteins may signal downstream along one or both pathways (Clapham and Neer, 1997). The G₅ isoform exhibits much less homology with the G_1-4 isoforms (~50%) and is preferentially expressed in neural tissue (Watson et al., 1994). A splice variant of G₅, G₅-long (G₅L), has been identified in retina that contains a 42 amino acid N-terminal extension (Watson et al., 1996).

When analyzed in vitro the G₅ isoform exhibits a degree of selectivity in its protein-protein interactions previously unknown among G subunits. Two categories of selective interaction have been documented for G₅ in vitro: G-dependent interactions with conventional G partners such as G subunits and G-responsive effectors (Zhang et al., 1996; Bayewitch et al., 1998; Fletcher et al., 1998; Lindorfer et al., 1998), and interactions with a subset of the regulator of G-protein signaling (RGS) proteins that appear to be G-independent (Snow et al., 1998, 1999; Levay et al., 1999; Makino et al., 1999).

The above in vitro findings leave open the important question of the functional associations of G₅ in brain. Cabrera et al. (1998) identified a native complex between G5 and RGS7 in a bovine retinal soluble fraction. However unlike in retina, where G₅ is almost entirely soluble, the majority of G₅ in the brain is membrane-associated (Watson et al., 1996). In this respect, it resembles conventional Gs in brain G complexes that are membrane-targeted by virtue of G subunit isoprenylation (Higgins and Casey, 1996). We describe here an approach to the purification G₅ from brain membranes, and under these experimental conditions, find no evidence of its association with several G or G subunits tested. Instead, brain membrane G₅ copurifies with the regulators of G-protein signaling, RGS6 and RGS7.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Brain membrane preparation and detergent extraction. Whole brains were collected from adult female CD1 mice and stored in liquid nitrogen. For each preparation, ~5 gm of mouse brain was thawed, minced, and homogenized in 50 ml of ice-cold homogenizing buffer on ice. Homogenization buffer consisted of 20 mM Na-HEPES, pH 7.4, 150 mM NaCl, 1 mM -mercaptoethanol, 3 mM MgCl₂, 17 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), 2 µg/ml of aprotinin, leupeptin, and pepstatin, and 1 µg/ml of soybean trypsin inhibitor. The homogenate was centrifuged at 1000 × g for 15 min to remove cell debris and followed by a centrifugation at 100,000 × g for 1 hr to precipitate the membranes. The membrane pellet was washed twice by resuspension and recentrifugation in homogenization buffer, then finally resuspended in 50 ml of solubilization buffer [homogenization buffer containing 0.2% w/v polyoxyethylene (10) monolauryl ether (Genapol C-100)] and stirred on ice for 2 hr. The detergent extract was centrifuged at 100,000 × g for 1 hr to remove insoluble particulate. The supernatant detergent extract was used immediately for immunoaffinity purification as described below.

Affinity purification of Ggb5 N-terminal antibody. Crude polyclonal antiserum ATDG was generated in rabbits against the synthetic 16-mer peptide ATDGLHENETLASLKS-amide, corresponding to residues 2-17 at the N terminus of mouse ₅ (Watson et al., 1994). Immobilized peptide beads were prepared by coupling the peptide to N-hydroxysuccinimide-activated agarose beads in 0.1 M 4-morpholinepropanesulfonic acid, pH 7.5 (Affi-Gel 15; Bio-Rad, Richmond, CA) and used to affinity purify anti-peptide antibody (Harlow and Lane, 1988).

Preparation of the ATDG antibody affinity column. Two methods were used: (1) to prepare ATDG antibodies covalently crosslinked to Protein A, 12.3 mg of Ig were crosslinked to 2 ml of Protein A agarose by dimethylpimelimidate in sodium borate buffer, pH 8.2 (Immunopure Protein A IgG Orientation kit; Pierce, Rockford, IL); (2) for noncovalent antibody-protein A columns, affinity-purified Igs (~15 mg) from rabbit ATDG antiserum were mixed with 2 ml of Protein A-coupled agarose beads pre-equilibrated with 500 mM NaCl/20 mM Tris-HCl, pH 7.5, (TBS) and incubated by end-over-end rotation at 4°C for 2 hr. The mixture was then split in half and after settling of the agarose beads, and each 1 ml column was equilibrated in solubilization buffer.

Affinity purification of the G5 complex and associated proteins. For an individual experiment, 50 ml of mouse brain detergent extract, prepared as described above, was applied to a 1 ml bed volume affinity column at the rate of ~2 ml/hr using a peristaltic pump in a 4°C cold room. Weakly bound proteins were washed off of the column by pumping 250 ml of the wash buffer [homogenization buffer containing 0.1% (w/v) Genapol C-100] through the column at the same rate as for the sample loading. After the last 6 ml of wash was examined by silver stain, the column was eluted with elution buffer [10 mM ATDG peptide in 0.1% (w/v) Genapol C-100; 20 mM Na-HEPES, pH 7.4, 150 mM NaCl, 1 mM -mercaptoethanol, 3 mM MgCl₂, 17 µg/ml AEBSF, 2 µg/ml of aprotinin, leupeptin, and pepstatin, and 1 µg/ml of soybean trypsin inhibitor]. The elution was performed by loading 1.5 ml of elution buffer onto the washed column and incubating for 24 hr at 4°C. The process was repeated until no detectable level of G₅ was present in the eluate (~ 5-6 elution fractions).

Gel electrophoresis and immunoblotting. Protein samples were separated on 4-20% gradient slab gels by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting according to standard procedures (Harlow and Lane, 1988). The primary antibodies used for immunoblotting were rabbit SGS polyclonal (Zhang et al., 1996), rabbit antiserum AS/7 (Goldsmith et al., 1987), rabbit antiserum QL (Shenker et al., 1991), rabbit EDPL polyclonal (Lee et al., 1995), rabbit anti-G₇ S-14 Ig (Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-RGS7 C-19 Ig (Santa Cruz). Antibodies were added at the dilution of 1:500 unless otherwise specified and incubated at room temperature for 2-16 hr. The blots were washed twice at 15 min intervals in TBS containing 0.2% (w/v) Tween 20. Secondary detection used [¹²⁵-I]Protein A (for rabbit antiserum) or [¹²⁵-I]Protein G (goat and other antiserum) followed by imaging on a storage phosphor screen. For visualization of protein bands, polyacrylamide gels were silver-stained and/or stained with Coomassie blue G250.

Identification of proteins associated with G5 protein. Aliquots of fractions collected off the affinity column with elution buffer were analyzed by SDS-PAGE and visualized by silver staining. Elution fractions containing the highest level of G₅ protein, as identified by Western blot analysis, were pooled and concentrated. The fractions were then separated by SDS-PAGE and visualized by staining with Coomassie blue G250. The broad band, whose elution profile paralleled that of the G₅ protein band in several elution fractions, was chosen as a candidate for further identification. The band of interest was excised from the gel, and tryptic peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight analysis (MALDI-ToF) (Borealis Biosciences Inc., Toronto, Ontario, Canada). Proteins were identified from the masses of the proteolytic peptides in the MALDI-ToF spectra by comparison with hypothetical proteolytic peptide masses derived from nonredundant translated genomic database using a Bayesian algorithm [ProFound version 3.2 software (Zhang and Chait, 1995)].

Immunoprecipitation. For each reaction, 1 ml of the above membrane extract of mouse brain was mixed with anti-G₅ or -RGS7 affinity-purified IgG or control IgG so that the final concentration of the IgG was ~40 nM and incubated at 4°C overnight. Then Protein A/G Plus Agarose beads (Santa Cruz Biochemicals) (15 µl) were added and incubated atroom temperature for 2 hr. At the end of incubation, the beads were pelleted by centrifugation at 10,000 × g for 2 min and washed with 3× 200 µl homogenization buffer described above. For specificity control reactions, blocking peptide (400 nM) were mixed with corresponding antibodies before the antibodies were mixed with membrane extracts. The proteins bound on the beads were released by adding 100 µl of 1× denaturing sample loading buffer and vortexing for 5 sec. Proteins were separated by SDS-PAGE and detected by Western blot analysis as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Purification of G₅ from mouse brain membranes

The concentration of G₅ in unfractionated mouse brain was estimated to be ~0.25% of total brain proteins by quantitative comparison of brain homogenates with a range of concentrations of highly purified recombinant G₅L on immunoblots developed with C-terminally directed G₅ antibody SGS (Zhang et al., 1996) and [¹²⁵-I] Protein A (data not shown). Of this total G₅ in mouse brain, 60% was membrane-associated and 40% was soluble (data not shown). This was in fair agreement with the findings of Watson et al. (1996), who found 70% of the G₅ in brain to be in the membrane pellet.

We chose the membrane fraction as a starting material for purification of G₅ from mouse brain. Because other G subunits in brain are membrane anchored through tight association with G subunits that are post-translationally lipid-modified by isoprenylation (Higgins and Casey, 1996), it was considered most likely that any G₅-G complexes in brain would be similarly expressed in the membrane fraction. Studies on purified recombinant G₅-G₂ complexes demonstrated an unusual sensitivity of G₅-G₂ interaction to bile salt-related detergents such as cholate and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid (Jones and Garrison, 1999). For this reason, brain membranes were extracted with the polyoxyethylene (10) monolauryl ether, Genapol C-100. Nonionic detergents of this type were found to be compatible with G₅-G₂ association in vitro (Jones and Garrison, 1999).

Detergent extraction and column-wise immunoaffinity purification of G₅ from brain membranes yielded ~30 µg of G₅ from 100 mg of protein in the starting brain membranes. The G₅ after this step ranged from ~10 to 30% pure, a degree of purity sufficient to allow clear resolution of G₅ at 39 kDa from other discrete protein bands by silver and Coomassie blue staining after SDS-PAGE (Fig. 2A).

Analysis of the flow through and eluate from the antibody column by immunoblotting documented the efficient capture of G₅ from the detergent extract and its release by the cognate peptide (Fig. 1). Of the total G₅ in the detergent extract applied to the antibody column, virtually all was retained, and ~25% could be recovered in the peptide eluate. Despite the concentration of G₅ by the antibody column, immunoblotting with antisera to several G and G subunits revealed no coelution with G₅ under these conditions (Fig. 1). Notably absent in the eluate were Gq and G₂, G-protein subunits shown in vitro to interact with G₅ (Fletcher et al., 1998).

View larger version (103K): [in this window] [in a new window]   Figure 1.   Analysis of G-protein subunits in mouse brain fractions by immunoblotting after anti-G5 immunoaffinity purification. Detergent extract from mouse brain was fractionated by passage over immobilized anti-G5 antibody and eluted with specific peptide, and the fractions were analyzed with antibodies to G-protein subunits, as described in Materials and Methods. The type of fraction is indicated above the lanes. In the left margin the antibody used in each immunoblot is indicated, and in the right margin the approximate Mr of the relevant immunoreactive band is indicated. The antibodies used include SGS against the C terminus of G5 (Zhang et al., 1996) (Anti-G5), antibody QL against the C terminus of Gq/11 (Shenker et al., 1991) (Anti-Gq/11), antiserum AS/7, which recognizes the C terminus of Gi1 and Gi2 (Goldsmith et al., 1987) (Anti-Gi1/2), polyclonal EDPL against the C terminus of G2 and G3 (Lee et al., 1995) (Anti-G2/3), and rabbit anti-G7 Ig S-14 (Anti-G7). In membrane detergent extract, flow through, and column wash lanes, 0.04% of sample was analyzed per gel lane and, in eluate lanes, 1.3% of total. The results from one purification experiment are shown, representative of three such experiments.

Proteins bound to the antibody column were eluted stepwise in the cold with aliquots of 10 mM peptide solution, and the resulting proteins were analyzed by staining after SDS-PAGE (Fig. 2). Several protein bands were seen whose elution profile bore no relation to the band at 39 kDa identified as G₅ by immunoblotting. One such band of 50 kDa was evident in silver-stained gels of the eluate fractions and peaked two fractions later than the G₅ band (at 39 kDa) (Fig. 2A,B). Other bands of slower mobility, which also bore no evident relation to the elution profile of G₅, were seen in preparative gels stained with Coomassie blue (Fig. 2C). Because of their lack of correlation with the elution of G₅, such bands were considered likely to represent nonspecifically bound proteins. A diffuse protein band at ~55 kDa was reproducibly found to elute in parallel to the G₅ at 39 kDa (Fig. 2A,B). The corresponding fractions of eluate were concentrated and run on a preparative SDS-PAGE, and the broad band at 55 kDa was excised from the gel for further analysis (Fig. 2C).

View larger version (80K): [in this window] [in a new window]   Figure 2.   Coelution of a 55 kDa protein band with G5. A, Consecutive peptide-eluted fractions (E1-E7) from the anti-G5 antibody column were analyzed by silver staining after separation by SDS-PAGE. Shown in the left margin are the relative mobilities of marker proteins (in kilodaltons) run in an adjacent lane (data not shown), and the position of the dye front (DF). Visible at 21.5 kDa in all lanes is soybean trypsin inhibitor included in the elution buffer. In the right margin is indicated the position of G5 at 39 kDa and the 55 kDa and 50 kDa bands as described in Results. B, Plot of the relative staining intensities of G5 at 39 kDa (filled diamonds), the 55 kDa band (open triangles), and the 50 kDa band (open squares) determined by densitometric analysis of the silver-stained gel shown in A. C, Preparative 4-20% SDS-PAGE gradient gel stained by Coomassie blue containing a concentrate of fraction E3 (Eluate 3). The mobility of marker proteins (in kilodaltons) in an adjacent lane, and the position of G5 at 39 kDa and the broad band at 55 kDa, which was excised for MALDI-ToF analysis, are indicated.

Identification of the 55 kDa protein band co-eluting with G5 as a mixture of RGS6 and RGS7

The 55 kDa protein band excised from the gel was trypsinized overnight, and the resulting tryptic peptides were subjected to MALDI-ToF mass spectroscopy. The masses of the proteolytic peptides in the MALDI-ToF spectra were compared with theoretical proteolytic peptide masses derived from the nonredundant translated genomic database using a Bayesian algorithm (Zhang and Chait, 1995) to allow identification of the component proteins. Peptides were identified that covered >50% each of the primary sequence of two proteins in the database, allowing identification of murine regulators of G-protein signaling RGS6 and RGS7 (Fig. 3). Within the 55 kDa protein band, assuming no sequence-specific bias in the recovery of peptides, the MALDI-ToF analysis of the recovered tryptic peptides suggested the two RGS proteins were present in approximately equimolar amounts.

View larger version (9K): [in this window] [in a new window]   Figure 3.   Coverage plots mapping tryptic peptides identified by mass spectroscopy from the 55 kDa protein band to the primary sequence of RGS6 and RGS7. A, The primary sequence of murine RGS6 is indicated by the top rectangle that is filled in black in positions corresponding to peptides identified by MALDI-ToF mass spectroscopic analysis, as described in Materials and Methods. The mass [and corresponding sequence (He et al., 1998) (GenBank accession number AF061933)] of the tryptic peptides identified include: 987.73 (K127-R134), 1134.77 (F302-K310), 1235.10 (S381-K391), 1317.89 (Q201-R211), 1446.32 (K200-R211), 1457.25 (F290-R301), 1506.69 (L83-R95), 1543.37 (Y357-K368), 1733.56 (A81-R95), 1755.45 (K103-K116), 1771.60 (I128-R141), 1797.22 (W270-R284), 1857.21 (Q201-K215), 1928.95 (D353-K368), 2500.98 (A135-R155), 2525.76 (S169-R191), 2654.45 (S169-K192), 2654.45 (K168-R191), and 3730.85 (R320-K352). B, The primary sequence of murine RGS7 is indicated by the top rectangle that is filled in black in positions corresponding to peptides identified by MALDI-ToF mass spectroscopic analysis, as described in Materials and Methods. The mass [and corresponding sequence (He et al., 1998) (GenBank accession number AF011360)] of the tryptic peptides identified include: 987.73 (K187-R194), 1120.71 (Y264-R271), 1120.71 (F362-K370), 1163.92 (S229-R238), 1276.14 (F362-R371), 1292.57 (K228 -R238), 1457.25 (F350-R361), 1479.44 (L143-R155), 1543.37 (Y417-K428), 1677.80 (W330-R344), 1706.63 (A141-R155), 1726.67 (K163-K176), 1771.60 (I188-R201), 1983.23 (E413-K428), 2413.91 (V381-K402), 2440.80 (A195-K215), 2836.19 (S239-K263), and 2983.13 (E376-K402).

Reciprocal coimmunoprecipitation of G₅ and RGS7 in unfractionated brain extract

To verify the presence of native complexes of G₅ and RGS7 in brain membranes, aliquots of an unfractionated detergent extract of mouse brain membranes were separately immunoprecipitated with antibodies to G₅ and RGS7 in the absence and presence of blocking peptide, or with control antibodies (Fig. 4). Comparable experiments with anti-RGS6 were not performed because of the lack of published or commercial sources for such an antibody. Immunoblots of the washed immunoprecipitates revealed the presence of both G₅ and RGS7 in the anti-G₅ pellets, and both were eliminated by the inclusion of specific blocking peptide (Fig. 4A,B). Similarly, both G₅ and RGS7 were present in the pellets of anti-RGS7 precipitates, and both were eliminated by the inclusion of the cognate RGS7 peptide (Fig. 4C,D). Neither G₅ nor RGS7 were present in immunoprecipitates using control rabbit or goat antibodies. Furthermore, immunoblot analysis of the same column fractions shown in Figure 1 with anti-RGS7 antibody demonstrated a concentration of RGS7 in the peak eluate fraction paralleling G₅ (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 4.   Reciprocal coimmunoprecipitation of G-protein 5 and RGS7 from unfractionated mouse brain extract. Aliquots of a Genapol C-100 detergent extract of whole mouse brain were immunoprecipitated (IP) with the indicated antibodies without or with an excess of antigenic peptide as shown, as described in Materials and Methods. Immunoblots with anti-G5 C-terminal antibody SGS (A, C) or anti-RGS7 C-terminal antibody (B, D) are shown, with the Mr of G5 (39 kDa) and RGS7 (55 kDa) indicated in the margins. Nonspecific bands at ~50 kDa in B and D correspond to the heavy chain of IgG. A, B, Immunoprecipitation with affinity-purified anti-G5 antibody ATDG or control rabbit IgG. C, D, Immunoprecipitation with anti-RGS7 IgG or control goat IgG.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Using an antibody-based purification scheme, native complexes of G₅ with RGS6 and RGS7 were isolated from mouse brain membranes. This is in line with the previous findings in retina in which native complexes between G₅ and RGS7 in the soluble fraction (Cabrera et al., 1998) and G₅L and RGS9 in the membrane fraction of rod outer segments (Makino et al., 1999) were identified. Mammalian RGS proteins 6, 7, 9, and 11 comprise a subset of the RGS proteins that contain a G-like domain that confers binding to G₅ and G₅L (Snow et al., 1998, 1999; Levay et al., 1999). This subset of RGS proteins shares a similar modular organization: an N-terminal dishevelled-Egl-10-pleckstrin (DEP) homology domain (Ponting and Bork, 1996) of unknown function, followed by a G-like domain, and finally the core RGS homology domain near the C terminus, known to function as a GTPase-activating protein (GAP) for heterotrimeric G subunits (Berman and Gilman, 1998). Because G₅ and G₅L appear to bind RGS proteins of this subfamily in 1:1 complexes to the exclusion of a G subunit (Levay et al., 1999; Makino et al., 1999), we presume that what we have isolated from brain represents a mixture of G₅-RGS6 and G₅-RGS7 complexes. We cannot rule out the presence of free G₅ in our isolate as well.

This is the first report in which RGS6 has been identified in a native protein complex. Expression of RGS6 mRNA has been documented by in situ hybridization in brain (Gold et al., 1997) and by Northern analysis in brain and heart (Snow et al., 1999). RGS6 and RGS7 are the most homologous pair (74% identical residues) among the four mammalian RGS proteins so far identified with DEP and G-like domains (Snow et al., 1999). The basis for membrane attachment of complexes of G₅ with RGS6 and RGS7 in brain remains obscure.

Although this purification scheme yielded ~25% of the G₅ from brain membranes, no associated G subunits could be detected in an immunological screen for several G subunits known to be neurally expressed. We cannot, of course, exclude the presence of other G subunits outside of our screen, or low levels of G below the limit of our detection method. The nonionic detergent used here was chosen based on its compatibility with G₅-G complex formation in vitro (Jones and Garrison, 1999). Nevertheless, associated G subunits may still have been lost during the relatively slow purification process described here, a methodology that may have favored isolation of high-affinity G₅-RGS complexes. Even if G₅ in brain was directly immunoprecipitated from detergent solution with the N-terminal antibody however, in place of the slower column-wise purification, no associated G subunits were detectable in blots of the immuno-precipitates (data not shown). Similarly, it is also possible the N-terminally directed anti-G₅ antibody selected against G₅-G complexes or actually promoted dissociation of G from G₅. Because G₅-G complexes have been shown in vitro to possess many functional properties expected of G (Zhang et al., 1996; Bayewitch et al., 1998; Fletcher et al., 1998; Lindorfer et al., 1998), an important remaining question is whether G₅-G complexes may exist transiently or constitutively within intact developing or adult neurons.

The present findings nevertheless suggest that in adult brain a large fraction of G₅ functions without an associated G subunit and therefore outside the classical conceptual framework of G interactions. The extent to which G₅-RGS complexes may function in a fashion analogous to G complexes is unknown. Similarly obscure is the role of the DEP domain and how the juxtaposition of an RGS core domain and G₅ within such complexes may mutually influence their protein-protein interactions. Answering the myriad questions raised by the discovery of G₅-RGS protein complexes will undoubtedly require future insights from many lines of experimental inquiry.

	FOOTNOTES

Received Oct. 19, 1999; revised Nov. 22, 1999; accepted Nov. 24, 1999.

The authors wish to thank Dr. Brian Cox of Borealis Biosciences for his expert help with the mass spectroscopy and Dr. Allen Spiegel for continuing support and encouragement. Correspondence should be addressed to Dr. William F. Simonds, National Institute of Diabetes and Digestive and Kidney Diseases, Metabolic Diseases Branch, Building 10, Room 8C-101, 10 Center Drive MSC 1752, Bethesda, MD 20892-1752. E-mail: wfs{at}helix.nih.gov.

This article is published in The Journal of Neuroscience, Rapid Communications Section, which publishes brief, peer-reviewed papers online, not in print. Rapid Communications are posted online approximately one month earlier than they would appear if printed. They are listed in the Table of Contents of the next open issue of JNeurosci. Cite this article as: JNeurosci, 2000, 20:RC59 (1-5). The publication date is the date of posting online at www.jneurosci.org.

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