JBC Avanti Polar Lipids

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M305764200 on September 30, 2003

J. Biol. Chem., Vol. 278, Issue 50, 50682-50690, December 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/50/50682    most recent
M305764200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Croci, C.
Right arrow Articles by Enz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Croci, C.
Right arrow Articles by Enz, R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Group I Metabotropic Glutamate Receptors Bind to Protein Phosphatase 1C

MAPPING AND MODELING OF INTERACTING SEQUENCES*

Cristina Croci{ddagger}, Heinrich Sticht{ddagger}, Johann Helmut Brandstätter§, and Ralf Enz{ddagger}

From the {ddagger}Emil-Fischer-Zentrum, Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, Erlangen 91054, Germany and §Max-Planck-Institut für Hirnforschung, Abteilung Neuroanatomie, Deutschordenstr. 46, Frankfurt 60528, Germany

Received for publication, June 2, 2003 , and in revised form, September 25, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The modulation of neurotransmitter receptors by kinases and phosphatases represents a key mechanism in controlling synaptic signal transduction. However, molecular determinants involved in the specific targeting and interactions of these enzymes are largely unknown. Here, we identified both catalytic {gamma}-isoforms of protein phosphatase 1C (PP1{gamma}1 and PP1{gamma}2) as binding partners of the group I metabotropic glutamate receptors type 1a, 5a, and 5b in yeast cells and pull-down assays, using recombinant and native protein preparations. The tissue distribution of interacting proteins was compared, and protein phosphatase 1C was detected in dendrites of retinal bipolar cells expressing the respective interacting glutamate receptors. We mapped interacting domains within binding partners and identified five amino acids in the intracellular C termini of the metabotropic glutamate receptors type 1a, 5a, 5b, and 7b being both necessary and sufficient to bind protein phosphatase 1C. Furthermore, we show a dose-dependent competition of these C termini in binding the enzyme. Based on our data, we investigated the structure of the identified amino acids bound to protein phosphatase 1C by homology-based molecular modeling. In summary, these results provide a molecular description of the interaction between protein phosphatase 1C and metabotropic glutamate receptors and thereby increase our understanding of glutamatergic signal transduction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The correct targeting and localization of proteins to synaptic specializations represents an important biological mechanism to regulate neuronal excitability. Increasing evidence underlines the importance of macromolecular signaling complexes, where functionally related proteins such as ion channels, neurotransmitters receptors, kinases, and phosphatases are arranged in close vicinity and are physically anchored to the synaptic cytoskeleton. Therefore, identification and characterization of interactions between synaptically localized proteins adds substantially to our understanding of molecular mechanisms of neurotransmission.

Receptors for glutamate are divided in ion channel-associated (ionotropic) receptors of the AMPA-, Kainate-, and NMDA-type and in G-protein coupled (metabotropic) receptors. Although ionotropic glutamate receptors mediate fast synaptic transmission, metabotropic glutamate receptors (mGluRs)1 modulate neuronal excitability and development, synaptic plasticity, transmitter release, and memory function using a variety of intracellular second messenger systems (1). The eight known members of this protein family are sub-divided into three groups, based on sequence homology, associated second messenger systems, and pharmacological properties (2). mGluR1 and mGluR5 (group I) stimulate phospholipase C, are selectively activated by quisqualate, and are generally expressed perisynaptically at postsynaptic sites (3–7). mGluR2 and mGluR3 (group II) are negatively coupled to adenylyl cyclase and do not show a specific preference for pre- or postsynaptic neurons (8–10). mGluR4, mGluR7, and mGluR8 (group III) are also negatively coupled to adenylyl cyclase and are primarily found at the active zone where they are suggested to function as glutamate auto-receptors (10, 11–16). Of the group III mGluRs the expression of mGluR6 is restricted to the retina, where it is localized postsynaptically at retinal photoreceptor to bipolar cell synapses, transmitting the "light on" signal in visual signal transduction (17, 18).

Glutamate-mediated neurotransmission depends critically on the regulation by associated proteins. The intracellular C termini of mGluR1 and mGluR5 (see Fig. 1A) interact with Calmodulin, Siah-1A, adenosine receptor A1, {alpha}-Tubulin, Tamalin, and Homer, the latter linking group I mGluRs to the NMDA/PSD-95 complex via Shank scaffold proteins (19–25). Isoforms of mGluR7 physically bind Filamin-A, {alpha}-Tubulin, PICK1, GRIP1, Calmodulin, Syntenin-1, and the {beta}{gamma}-subunit of G-proteins (26–30). Recently, we identified two catalytic isoforms of protein phosphatase 1C (PP1C), PP1{gamma}1 and PP1{gamma}2, as binding partners of mGluR7b (31). No interaction was observed with the mGluR7a isoform, indicating that binding of PP1C to mGluR7b was regulated by alternative splicing of the mGluR7 transcript. Furthermore, preliminary results of our laboratory indicated a physical interaction between PP1{gamma}1 and mGluR5 in yeast cells.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1.
PP1{gamma}1 interacts with C-terminal domains of mGluRs. A, left, domain structure of mGluRs. The membrane is indicated by a gray box, black rectangles represent transmembrane regions 1–7, and the intracellular C terminus is drawn in bold. Right, GST and GST fusion proteins were immobilized on glutathione-Sepharose and incubated with T7-tagged PP1{gamma}1 purified from E. coli as indicated or soluble native protein extracts prepared from rat brain. Bound proteins were detected on Western blots using a monoclonal anti-T7 immune serum (upper panel) or specific antibodies for PP1{gamma}1 or PKC-{zeta}. Calculated sizes of PP1{gamma}1 and PKC-{zeta} are indicated on the left in kDa. Protein concentrations of coated Sepharose beads are shown on Coomassie-stained SDS-PAGE (arrowheads in lower panel), with the Benchmark prestained protein ladder (Invitrogen) in the left lane. B, mGluR1a, mGluR5a/b, and PP1{gamma}1 were detected by their specific antibodies in microsomal membrane proteins prepared from rat brain (left panel). Immunoprecipitations were performed with the immunesera indicated, and bound PP1{gamma}1 was detected on Western blots (right panel). The Benchmark prestained protein ladder is indicated on the left in kDa.

 
Here, we investigated the binding between the two PP1C isoforms PP1{gamma}1 and PP1{gamma}2 and the group I metabotropic glutamate receptors mGluR1 and mGluR5. We compared the expression profile between PP1{gamma}1/2 and interacting mGluRs and showed in the retina that PP1{gamma}1 is present in the same subcellular compartments as mGluR1 and mGluR5. Furthermore, we mapped PP1{gamma}1-binding amino acids in the interacting glutamate receptors mGluR1a, mGluR5a/b, and mGluR7b and investigated their three-dimensional structure using homology-based molecular modeling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
All animal experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany, the National Institutes of Health, and the Max Planck Society.

Molecular Biology—Domains of mGluR C termini were PCR-amplified from rat brain cDNA and sub-cloned in the yeast two-hybrid bait vector pBTM116 or fused to the coding sequence of glutathione S-transferase (GST) in pET41 (Novagen, Madison, WI). The complete coding regions of rat PP1{gamma}1 and PP1{gamma}2 were also generated by PCR, ligated in the yeast two-hybrid prey vector pVP16, or fused to the T7-epitope of pET21 (Novagen). Mutation and deletion constructs of mGluR C termini and PP1{gamma}1/2 were generated by PCR cloning techniques. All constructs were sequenced to check for PCR errors.

Binding Assays—For GST pull-down assays pET21 and pET41 constructs were transformed in Escherichia coli BL21(DE3)pLysS, and protein expression was induced by adding 1 mM isopropyl-beta-D-thiogalactoside (Sigma-Aldrich). Fusion proteins were purified under native conditions, immobilized to glutathione-Sepharose beads, and incubated with the cytosolic fractions of E. coli expressing PP1{gamma}1 as described (31, 32). C-terminal domains of mGluR1a, -5a, and -5b were also synthesized in vitro using the T7 promoter of pET41 according to the RiboMAX RNA Production Kit and the Flexi Rabbit Reticulocyte Lysate System (Promega) in the presence of [35S]methionine (Amersham Biosciences). To obtain comparable conditions in competition experiments, E. coli protein extracts of similar protein concentrations, as measured at 280 nm, were used. For these samples the total volume was adjusted to 400 µl (defined as 100%) by adding protein extract of non-transformed E. coli BL21(DE3)pLysS. Bound proteins were separated on SDS-PAGE and visualized with Coomassie Brilliant Blue R-250 (Serva, Heidelberg, Germany) or detected by Western blotting using a monoclonal anti-T7 antibody and the enhanced chemiluminescence system (ECL; Amersham Biosciences). Unless otherwise stated all reagents were purchased from Novagen.

For binding assays with native proteins, rat brains were homogenized in 0.32 M Saccharose, 20 mM Tris-HCl, pH 7.4, containing DNaseI and protease inhibitors (Roche Diagnostics) and centrifuged at 700 x g for 5 min as described (32). The supernatant was collected and centrifuged at 30,000 x g for 30 min, and 5 mg of the resulting supernatant were incubated with glutathione-Sepharose beads, coated with GST or GST fusion proteins, for 5 h under slow agitation. Interaction partners were analyzed on Western blots using specific immunesera for PP1{gamma}1 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and PKC-{zeta} (1:10000; Sigma-Aldrich).

For immunoprecipitation assays, rat brain microsomal membrane proteins (Upstate Biotechnology, Lake Placid, NY) were incubated for 3 h at 4 °C with antibodies specific for mGluR1a (5 µg; Upstate Biotechnology), mGluR5a/b (6 µg; Upstate Biotechnology), PP1{gamma}1 (15 µl; kindly provided by Paul Greengard, Rockefeller University, New York; see Ref. 33), or PKC-{zeta} (5 µl; Sigma-Aldrich). Then 50 µl of a 50% (v/v) suspension of protein A-Agarose (Amersham Biosciences) were added, and the mixture was incubated overnight as above. Precipitates were washed three times with 500 µl of 150 mM NaCl, 0.1% Triton X-100, 10% glycerin, 50 mM HEPES, pH 7.5, containing protease inhibitors (Roche Diagnostics), and bound PP1{gamma}1 was detected on Western blots using its specific immune serum (1:1000; see Ref. 33).

Reverse Transcriptase PCR—Total RNA was extracted from adult rat tissues and reverse transcribed as described (32). PCR amplification was performed with 150 ng of reverse-transcribed RNA using specific primers for PP1{gamma}1+2 (sense nt 677–698; antisense PP1{gamma}1 nt 972–948; antisense PP1{gamma}2 nt 1014–993), mGluR1a (sense nt 2521–2540; antisense nt 2796–2776), and mGluR5a/b (sense nt 2479–2498; antisense nt2753–2734) in a programmable thermocycler (Applied Biosystems, Foster City, CA) with the following parameters: 94 °C, 2 min; 30 cycles at 94 °C, 45 s; 62 °C, 60 s; 72 °C, 30 s; and a final incubation at 72 °C, 10 min. Oligonucleotides recognizing {beta}-actin (sense nt 372–391; antisense nt 1065–1046) were used as controls, using 25 PCR cycles with the same parameters. Additional controls were performed without adding template and/or reverse transcriptase and showed no amplified DNA. Five µl of each PCR reaction were separated on 1% agarose gels and detected on Southern blots using digoxygenindidesoxy-UTP (DIG-ddUTP)-labeled oligonucleotides specific for PP1{gamma}1/2 (nt 807–786), mGluR1a (nt 2758–2766), mGluR5a/b (nt 2626–2646), and {beta}-actin (nt 732–750) following the protocol of the DIG-Oligonucleotide Tailing Kit and the DIG Luminescent Detection Kit (Roche Diagnostics).

Immunocytochemistry—Adult mice were deeply anesthetized with halothane and decapitated. Retinal cryostat sections with a thickness of 12 µm were prepared for immunocytochemical labeling by the indirect fluorescence method as described (14). The retinal sections were incubated with primary antibodies directed against PKC-{alpha} (1:100; Dunn Labortechnik, Asbach, Germany) and PP1{gamma}1 (1:1000; kindly provided by Paul Greengard, Rockefeller University, New York; see Ref. 33) overnight at room temperature. Binding of primary antibodies was detected by goat anti-mouse or goat anti-rabbit IgG secondary antibodies coupled to AlexaTM 594 (red fluorescence) or AlexaTM 488 (green fluorescence) (1:500; Molecular Probes, Eugene, OR). In control experiments, either the primary or secondary antibody was omitted, resulting in a complete loss of specific immunoreactivity. Sections were examined with a Zeiss confocal laser scanning microscope equipped with argon and HeNe lasers (LSM5 Pascal; Zeiss, Oberkochen, Germany).

Yeast Two-hybrid Techniques—Protein interactions were tested in the yeast strain L40 (Invitrogen) as described (31). In brief, yeast strains expressing bait constructs were transformed with prey constructs and vice versa, and protein interactions were monitored by the activation of His3 and LacZ reporter genes on selection plates. Binding affinities were calculated according to the Yeast Protocols Handbook (Clontech, Palo Alto, CA) using o-nitrophenyl-{beta}-D-galactopyranoside (Sigma-Aldrich) as substrate.

Comparative Modeling—The crystal structure of PP1{gamma}1 in complex with a RRVSFA peptide (34) was used as template to model PP1{gamma}1 binding sequences (KSVSWS, KSVTWA, KSVTWY) identified in this study. Models were generated by substituting side chains of the RRVSFA ligand using the Sybyl 6.5 program package (Tripos, Inc.). Noncovalent interactions were improved by 200 steps of conjugate gradient energy minimization using the Powell algorithm (35). The stereochemistry and nonbonded contacts were evaluated using the programs PROCHECK (36) and WHAT_CHECK (37). Protein-ligand contacts were analyzed with LIGPLOT (38) using default settings.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PP1{gamma}1 Binds to mGluR1a and mGluR5a/b—Preliminary experiments of our laboratory indicated binding of PP1{gamma}1 to mGluR5 in yeast cells. Therefore, interactions between PP1{gamma}1 and the group I metabotropic glutamate receptors mGluR1a, mGluR5a, and mGluR5b were now tested in GST pull-down assays using immobilized C-terminal domains of mGluR1a, mGluR5a, and mGluR5b C termini fused to GST. Loaded glutathione-Sepharose beads were incubated with E. coli protein extracts or soluble native proteins from rat brain, and bound PP1{gamma}1 was identified by Western blots using specific antibodies. Both recombinant and native PP1{gamma}1 interacted with mGluR1a, mGluR5a, and mGluR5b (Fig. 1A). The previously described interaction of PP1{gamma}1 with mGluR7b, and absence of interaction with mGluR7a (31), served as positive and negative controls, respectively.

To ensure that GST fusion proteins did not unspecifically bind to proteins of the extracts, we analyzed their interaction with the {zeta}-isoform of protein kinase C (PKC-{zeta}). This enzyme was present in the soluble protein fraction of rat brain (32), and a specific immune serum was available in the laboratory. Interaction between PKC-{zeta} and metabotropic glutamate receptors has not been reported, and consistently we did not detect its binding to mGluR C termini (Fig. 1A).

Next, we tested whether the observed interactions between PP1{gamma}1 and mGluR1a and mGluR5a/b C-terminal domains also occurred with the native forms of the metabotropic glutamate receptors. As a source for mGluR1a and the two mGluR5 isoforms we used microsomal membrane proteins purified from rat brain that also contained high concentrations of PP1{gamma}1 (Fig. 1B, left panel). Immunoprecipitation experiments with specific antibodies for mGluR1a and mGluR5a/b demonstrated binding of PP1{gamma}1 to both receptor types (Fig. 1B, right panel). Antibodies against PP1{gamma}1 and PKC-{zeta} were used as positive and negative controls, respectively.

Tissue Distribution of mGluR1a, mGluR5a/b, and PP1{gamma}1/2— Next, we compared the expression profiles of PP1{gamma}1, its isoform PP1{gamma}2, and of mGluR1a, mGluR5a, and mGluR5b performing semi-quantitative reverse transcriptase-PCR. To reduce variability between samples, the same primer pair was used to amplify both splice variants of mGluR5. Furthermore, DNA products representing mGluR5a and mGluR5b, as well as PP1{gamma}1 and PP1{gamma}2, were visualized using an oligonucleotide hybridizing to both isoforms.

mGluR1a and mGluR5 isoforms were present in all regions of the central nervous system (CNS) examined, although in different amounts (Fig. 2). The isoform distribution between mGluR5a and mGluR5b varied between CNS regions. mGluR5a was predominantly expressed in olfactory bulb and cerebellum, whereas mGluR5b was higher concentrated in cortex, hippocampus, and thalamus. PP1{gamma}1 was present in all CNS regions examined, whereas transcripts for PP1{gamma}2 were more diversely expressed, being most abundant in cerebellum (Fig. 2). Outside the CNS, PP1{gamma}1, and PP1{gamma}2 seemed to be ubiquitously expressed, whereas only mGluR1a was detected in lung.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 2.
Expression profiles of PP1{gamma} isoforms and interacting mGluRs. Southern Blots of PCR products for mGluR1a, mGluR5a/b, PP1{gamma}1, and PP1{gamma}2, obtained after reverse transcription of total RNA isolated from the indicated tissues, are shown. Amplification of fragments for {beta}-actin compared cDNA concentrations between samples. Control reactions without addition of reverse transcriptase resulted in absence of PCR products (not shown).

 
PP1{gamma}1 Is Present at Retinal Synapses Expressing mGluR1a and mGluR5a/b—Co-expression of binding partners represents a pre-requisite for protein interactions. In the retina, the localization of mGluR1a and mGluR5a/b was analyzed in detail (39). Therefore, we tested whether PP1{gamma}1 is present in the same sub-cellular compartments as described for mGluR1a and mGluR5a/b. Staining of vertical sections of adult mouse retinae with an antiserum against PP1{gamma}1 showed expression of PP1{gamma}1 in the two synaptic layers of the retina, the outer (OPL) and inner (IPL) plexiform layer (Fig. 3A). No staining for PP1{gamma}1 was found on somata of interneurons in the inner nuclear layer (INL) and of ganglion cells in the ganglion cell layer (GCL). In the IPL, PP1{gamma}1 immunoreactivity was homogeneously distributed in small immunofluorescent puncta, suggestive of a synaptic localization of the enzyme. In the OPL, small immunofluorescent puncta (arrows in Fig. 3A) were visible above larger aggregates of PP1{gamma}1 staining (arrowheads). In this layer, the small synaptic terminals of rod photoreceptors are located above the large cone photoreceptor synaptic terminals. Thus, the small puncta indicated PP1{gamma}1 expression at rod synapses, whereas the larger immunoreactive aggregates suggested expression of PP1{gamma}1 in neurons postsynaptic to cone photoreceptors.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 3.
Synaptic localization of PP1{gamma}1 in the retina. Confocal micrographs of a vertical retinal section double-labeled for PP1{gamma}1 (green) and PKC-{alpha} (red) as a marker for rod bipolar cells are shown. A, PP1{gamma}1 is expressed in the OPL and IPL but not in somata of interneurons in the INL and of ganglion cells in the GCL. The localization of PP1{gamma}1 in the OPL suggested its expression post-synaptic to rod (arrows) or cone (arrowheads) photoreceptors. B, PP1{gamma}1 is co-localized with the dendrites of rod bipolar cells (arrows). Higher power views of the labeling for PP1{gamma}1 (C) and rod bipolar cells (D) in the OPL are shown. In D, the somata of the rod bipolar cells and their dendrites, which extend toward the rod photoreceptor synaptic terminals, can be seen. E, the merge of the stainings for PP1{gamma}1 (green) and for rod bipolar cells (red), as shown in enlargements of the selected boxes in C and D, clearly demonstrates the localization of PP1{gamma}1 in rod bipolar cell dendrites (yellow). Scale bars are 10 µm.

 
Group I mGluRs are expressed by the postsynaptic neurons of photoreceptors, especially in the dendrites of rod bipolar cells postsynaptic to the rod photoreceptor synaptic terminals (39). Therefore, sections were double-labeled with antibodies against PP1{gamma}1 (green) and against PKC-{alpha} (red), a marker for rod bipolar cells (Fig. 3B). The rod bipolar cell somata are located in the INL, their dendrites extend into the OPL toward the synaptic terminals of the rod photoreceptors, and their axons terminate in the innermost part of the IPL close to the GCL. Comparison of the staining for PP1{gamma}1 in the OPL with the dendritic structures of the labeled rod bipolar cells by confocal laser-scanning microscopy unambiguously demonstrated that PP1{gamma}1 was expressed in the dendrites of rod bipolar cells (Fig. 3, C and D). This is best seen in Fig. 3E, showing higher power views of the merge of the two stainings for selected areas (boxes) in the OPL.

Mapping of Interacting Domains within mGluR C Termini and PP1{gamma}1/2—Based on our findings, we proposed the existence of PP1{gamma}1/2 binding motifs located in the C-terminal domains of mGluR1a, mGluR5a, and mGluR5b. To map interacting domains within the binding partners, we analyzed the interaction between PP1{gamma}1 and mGluR C termini in yeast cells by the ability of transformants to grow on selective media upon activation of His3 and LacZ reporter genes. The complete C termini of mGluR1a, mGluR5a, and mGluR5b showed transactivation of the reporter genes, which had been also noticed by other groups (21, 25). Therefore, we used C-terminally truncated constructs for our studies (see stars in Fig. 4A) that were able to bind recombinant and native PP1{gamma}1 in GST pull-down assays (data not shown). In yeast cells PP1{gamma}1 interacted with mGluR1a*, mGluR5a*, and mGluR5b*, whereas no binding was observed for mGluR1b and mGluR1c (Fig. 4A). The PP1{gamma}1 binding characteristics of mGluR7a and mGluR7b have been reported before (31) and were used as controls for the assay. The relative intensities of the interactions were estimated using a quantitative {beta}-galactosidase assay and are presented as arbitrary {beta}-galactosidase units (horizontal columns in Fig. 4A).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4.
Domain mapping of interaction partners. Complete C termini of mGluR1, mGluR5, and mGluR7 isoforms (A) or their proximal or distal regions (B) were individually tested in yeast cells for their ability to bind PP1{gamma}1, as indicated. Interaction was monitored by the growth of transformants on selective media upon activation of the His3 and lacZ reporter genes, indicated by + or -; t, transactivation of reporter genes. Stars refer to truncated C termini used because of transactivation of the complete domains. Encoded amino acids are represented by numbers in parentheses. Relative affinities were quantified and visualized as arbitrary {beta}-galactosidase units ({beta}-Gal; horizontal columns). Each value represents the mean of three to six yeast clones. Error bars are ± S.E. C, a series of deletion constructs of PP1{gamma}1 and PP1{gamma}2 were tested for their binding to C-terminal sequences of mGluR1a, mGluR5a, and mGluR5b as in A. Domains analyzed of PP1{gamma}1/2 are indicated as follows: NT, N terminus; core, core region; D, C-terminal deletion tolerated for functional expression of the enzyme; S, splice site.

 
To identify PP1{gamma}1 binding regions within interacting mGluR C termini, their identical and splice-specific domains were tested individually. The proximal region of the mGluR1a C terminus, identical to the corresponding regions in mGluR1b and mGluR1c, showed no interaction with PP1{gamma}1, whereas the distal, isoform-specific sequence of mGluR1a* (mGluR1a*sp) was sufficient for the binding (Fig. 4B). In contrast, the isoform-specific cassette inserted into the C terminus of mGluR5b did not interact with PP1{gamma}1, whereas a region identical between mGluR5a and mGluR5b (mGluR5*sp) contained enough information for the binding. Again, reported interactions of mGluR7a and mGluR7b C-terminal domains were used as positive and negative controls. From these results we concluded that the PP1{gamma}1 binding motif is located in the splice-specific distal region of mGluR1a and mGluR7b, whereas the isoform-specific sequence of mGluR5b does not seem to be involved in the binding.

We also mapped domains in PP1{gamma}1 and PP1{gamma}2 mediating the binding to mGluR1a and mGluR5a/b C termini, following a classification of functional domains described for PP1{alpha} (40). PP1{gamma}1/2 was divided in the N terminus, a conserved core region, and the C-terminal part that was further sub-divided into a region needed for functional expression of PP1{alpha} (residues 270–297; see Ref. 41), the part proximal of the splice site, and the two distal domains different between PP1{gamma}1 and PP1{gamma}2 (Fig. 4C).

N- and C-terminal deletion constructs of PP1{gamma}1/2 showed identical binding characteristics for all three tested mGluR C termini (Fig. 4C). Deletion of 40 N-terminal amino acids abolished interactions, whereas removal of the isoform specific C termini of PP1{gamma}1/2 had no effect. C-terminal deletions upstream of position 297 prevented binding, consistent with the result of Zhang et al. (41), who demonstrated that amino acids 1–297 were needed for functional expression of the protein. The calculated relative binding intensities are shown for the minimal interacting PP1{gamma}1/2 region, represented by construct P-b, (vertical columns in Fig. 4C), and were similar to the full-length PP1{gamma}1 (see Fig. 4A), indicating that regions C-terminal of amino acid 297 do not participate in the interactions.

Identification of PP1{gamma}1 Binding Motifs in Interacting mGluR C Termini—To map PP1{gamma}1 binding motifs in mGluR C termini, we compared the amino acid sequences of the minimal interacting C-terminal domains of mGluR1a, mGluR5a/b, and mGluR7b, as determined in Fig. 4B. This alignment revealed one region of high similarity, ranging from position 888 to 895 of mGluR1a (Fig. 5A). It seemed unlikely that Ser-888 takes part in the PP1{gamma}1 binding motifs, because its deletion in mGluR7b did not alter the binding to PP1{gamma}1 (31). Thus, we proposed PP1{gamma}1 binding motifs consisting of the amino acids KSVSW for mGluR1a and of the sequence KSVTW for both mGluR5 isoforms and for mGluR7b (bold characters in Fig. 5A).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5.
Identification of PP1{gamma}1 binding motifs in mGluR C termini. A, alignment of PP1{gamma}1 interacting protein regions of mGluR1a, mGluR5, and mGluR7b. The proposed PP1{gamma}1 binding motifs are shown in bold. B–D, deletion constructs of mGluR1a, mGluR5, and mGluR7b were analyzed for binding PP1{gamma}1 in yeast cells, and results were verified using GST pull-down experiments (E). Interactions were analyzed and quantified as in Figs. 1 and 4. For clarity, only 14 residues of the tested protein sequences are shown in B–D, and the actually tested regions are indicated by numbers in parentheses of the wild type (WT) domains.

 
We tested this hypothesis by examining a series of deletion and mutant constructs for their ability to bind PP1{gamma}1 in yeast cells and GST pull-down assays. First, we deleted the proposed PP1{gamma}1 binding motif, as well as left and right flanking sequences. Consistent with our hypothesis, residues N- or C-terminal to the binding motif had no effect on the interactions (Fig. 5, B–D). In contrast, deleting the five amino acids of the proposed binding motifs completely abolished the interaction with PP1{gamma}1. The estimated binding affinities of the mutated constructs were similar to those observed for the wild type sequences (horizontal columns in Fig. 5, B–D), except for mGluR7b-sp{Delta}YTI, probably because of two prolines adjacent to the PP1{gamma}1 binding motif. We verified the observed binding characteristics of the deletion mutants in GST pull-down experiments (Fig. 5E) and concluded that the amino acid sequences KSVSW and KSVTW contributed substantially to the PP1{gamma}1 binding motifs in mGluR C termini.

To assess the contribution of each individual residue of these motifs for the protein interactions, each amino acid of the PP1{gamma}1 binding motifs was replaced independently by alanine. Except for serine at position +2 of the motifs, all mutants failed to interact with PP1{gamma}1, indicating that amino acid side chains, rather than the protein backbone forms the PP1{gamma}1 binding surface (Fig. 6). Although the hydroxyl group of serine at position +2 seemed not to participate in the binding surface contacting PP1{gamma}1, an amino acid at this position might be needed to space the neighboring interacting residues correctly. To test this hypothesis, we deleted this serine in all three mGluR C termini, which prevented their binding to PP1{gamma}1 (Fig. 6). Therefore, an amino acid at this position, although not directly needed to interact with PP1{gamma}1, might be required for the correct three-dimensional structure of the PP1{gamma}1 binding motif.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6.
Alanine scan of PP1{gamma}1 binding motifs in mGluR C termini. Amino acids forming the proposed PP1{gamma}1 binding motifs of mGluR1a (A), mGluR5 (B), and mGluR7b (C) are shown in bold and were independently substituted with alanine or deleted. The ability of generated constructs to interact with PP1{gamma}1 was analyzed in yeast cells as in Fig. 4. As in Fig. 5, only parts of the tested protein sequences, given in parentheses, are shown for clarity. WT, wild type.

 
Identified Protein Phosphatase Binding Motifs Are Necessary and Sufficient to Interact with PP1{gamma}1—To analyze whether the identified five amino acids in the mGluR1a, mGluR5a/b, and mGluR7b C termini are indeed sufficient to bind PP1{gamma}1, we introduced their binding motifs into the C terminus of mGluR7a that did not interact with PP1{gamma}1 (see Figs. 1 and 4). The sequences KSVSW and KSVTW were inserted in mGluR7a at a position corresponding to the location of the mGluR7b motif (Fig. 7A). Although the wild type sequence of mGluR7a-sp did not bind PP1{gamma}1, mGluR7a-sp carrying the motifs KSVSW and KSVTW interacted in yeast cells (Fig. 7A) and in GST pull-down assays (Fig. 7B). Thus, the identified PP1{gamma}1 binding motifs are necessary and sufficient for the protein interactions.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 7.
Identified binding motifs in mGluR C termini are sufficient to interact with PP1{gamma}1. A, alignment of the splice specific C-terminal domains of mGluR7a and mGluR7b. The proposed PP1{gamma}1 binding motif in mGluR7b is shown in bold. PP1{gamma}1 binding motifs of mGluR1a (KSVSW) and mGluR5/mGluR7b (KSVTW) were introduced into mGluR7a, and mutants were analyzed for their ability to interact with PP1{gamma}1 in yeast cells (A) and GST pull-down experiments (B), as described in Figs. 1 and 4.

 
Next, we used the C terminus of mGluR7b, containing the identified KSVTW motif, to compete the binding of PP1{gamma}1 with group I mGluRs. First, PP1{gamma}1 was bound to the C termini of mGluR1a, mGluR5a, and mGluR5b immobilized on glutathione-Sepharose beads (Fig. 8, A-C, rows 1). These beads were then incubated with increasing concentrations of the His-tagged C terminus of mGluR7b containing the KSVTW motif, or with His-mGluR7a, not interacting with PP1{gamma}1 as control (Fig. 8, A–C, rows 2). mGluR7b competed with immobilized mGluR1a*, mGluR5a*, and mGluR5b* for the binding to PP1{gamma}1 and displaced the enzyme from the coated glutathione-Sepharose beads into the supernatant. The remaining amount of PP1{gamma}1 bound to mGluR1a*, mGluR5a*, and mGluR5b* was analyzed by separating the coated glutathione-Sepharose beads from the supernatant and subsequent detection of PP1{gamma}1 on Western blots (Fig. 8, A–C, rows 3). Displaced PP1{gamma}1, bound to His-mGluR7b in the supernatant, was analyzed on Western blots after pulling down the proteins using Ni-NTA beads (Fig. 8, A–C, rows 4). We also controlled that PP1{gamma}1 itself did not bind to GST- or Ni-NTA beads, and that the His-tagged mGluR7 C termini did not interact with the glutathione-Sepharose beads (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 8.
Identified binding motifs in mGluR C termini compete for PP1{gamma}1. PP1{gamma}1 was bound to the C termini of mGluR1a (A), mGluR5a (B), and mGluR5b (B) immobilized on glutathione-Sepharose (rows 1). Identical amounts of beads were then incubated with increasing concentrations of His-mGluR7b, containing the KSVTW motif, or with His-mGluR7a for control (rows 2). Competition among mGluR1a*, mGluR5a*, mGluR5b*, and mGluR7b, but not mGluR7a, displaced PP1{gamma}1 from the coated glutathione-Sepharose beads into the supernatant. Remaining amounts of PP1{gamma}1 bound to mGluR1a*, mGluR5a*, and mGluR5b* were analyzed by pulling down the glutathione-Sepharose beads and subsequent detection of PP1{gamma}1 on Western blots, as described in Fig. 1 (rows 3). Displaced PP1{gamma}1 interacted with HismGluR7b, but not with His-mGluR7a, and was detected on Western blots using Ni-NTA beads (rows 4). The different pull-downs of PP1{gamma}1 are indicated by the names of the GST or His fusion proteins in parentheses. Protein concentrations of GST- and His-tagged fusion proteins are shown on Coomassie-stained SDS-PAGE (arrowheads).

 
Increasing concentrations of mGluR7b were able to compete with mGluR1a, mGluR5a, and mGluR5b for interaction with PP1{gamma}1 in a dose-dependent manner, whereas mGluR7a had no effect (Fig. 8). Because the amino acid sequence KSVTW of mGluR7b is present in mGluR5a/b and homologous to the sequence KSVSW of mGluR1a (see Fig. 5A), these results strongly indicate an important contribution of these motifs in PP1{gamma}1 binding.

Comparative Modeling of the Identified PP1{gamma}1 Binding Motifs—Co-crystallization of PP1{gamma}1 with the peptide RRVSFA, corresponding to amino acids 64–69 of the PP1C regulatory G-subunit (GM), demonstrated the formation of a {beta}-strand comprising residues 67–69 of GM that is incorporated into a {beta}-sheet of PP1C (34). This sequence is similar to the described PP1{gamma}1/2 binding motifs KSVSW and KSVTW located in mGluR C termini. To obtain a three-dimensional representation of the interaction between mGluR C termini and PP1{gamma}1, we modeled the identified PP1{gamma}1 binding motifs in a complex with the enzyme, based on the reported structure of the PP1{gamma}1/GM complex (34). Because this study analyzed the structure of six amino acids of GM (RRVSFA) bound to PP1{gamma}1, we included the analogous sixth position of the mGluR sequences (see Fig. 5) in our modeling.

Inspection of the modeled complexes among mGluR1a, mGluR5a/b, mGluR7b, and PP1{gamma}1 revealed that side chain substitutions and subsequent energy minimizations only resulted in very minor structural changes compared with the PP1{gamma}1/GM interaction (Fig. 9, A-E). Analysis of the stereochemistry and nonbonded contacts confirmed that even for the sterically most demanding PP1{gamma}1 binding motif KSVTWY, carrying a tyrosine instead of an alanine at position +6, no steric strain was imposed on the complex by the modeling procedure, and all specific side chain interactions reported for the RRVSFA peptide (34) were preserved (Fig. 9, E and F).



View larger version (84K):
[in this window]
[in a new window]
  		FIG. 9.
Proposed structure of identified PP1{gamma}1 binding motifs. A, solvent accessible surface area and electrostatic potential of PP1{gamma}1 in complex with the RRVSFA peptide of the regulatory G-subunit or with the mGluR C-terminal sequences KSVSWS (B), KSVTWA (C), and KSVTWY (D). Peptides are shown in stick representation, and the protein surface is colored according to the electrostatic potential (red, most negative; blue, most positive). Coordinates in A were taken from Ref. 33. E, overlay of the RRVSFA template (red) with the sterically most demanding KSVTWY sequence (green). The solvent accessible surface area of PP1{gamma}1 is shown in white. F, schematic representation of the contacts between PP1{gamma}1 and the KSVTWY sequence. The peptide is shown in ball-and-stick representation with carbon, oxygen, nitrogen, and sulfur atoms colored in black, red, blue, and yellow, respectively. Bonds of the protein backbone are shown in black, and those of the side chains in blue. Green dotted lines indicate polar interactions between the peptide and PP1{gamma}1, with their distance given in Angstroms. The corresponding residues of PP1{gamma}1 are highlighted by gray rectangles. Hydrophobic interactions are indicated by red dashes for peptide atoms and red circles for the corresponding residues of PP1{gamma}1.

 
At the first position of the binding motifs all modeled peptides contain a lysine, capable of forming a hydrogen bond to the carboxyl group of the PP1{gamma}1 residue Glu-287 (Fig. 9F), similar to the corresponding arginine in the crystallized PP1{gamma}1/GM complex (34). The amino acid present at the second position showed neither in the reported crystal structure nor in the modeled peptides any specific side chain interactions (Fig. 9), consistent with our experimental data in which this residue could be replaced by alanine (Fig. 6). The observation that this position tolerated a high sequence variability but no deletion (Fig. 6) could be explained by the extended backbone conformation of the peptide forcing the side chains of adjacent residues to point into opposite directions. Deletion of the second residue in the PP1{gamma}1 binding motifs would disrupt this pattern resulting in the loss of interactions formed by lysine and valine residues in position +1 and +3 (Fig. 9F).

The side chains of the two hydrophobic residues of the motifs, valine and tryptophan at positions +3 and +5, were directed toward the PP1{gamma}1 surface and formed numerous hydrophobic contacts (Fig. 9, E and F). The binding site of valine was mainly formed by the PP1{gamma}1 side chains of Ile-169, Leu-243, Leu-289, and Cys-291, whereas that for tryptophan was formed by side chains of Phe-257, Met-283, Cys-291, and Phe-293. Interestingly, the latter binding pocket that was occupied by a phenylalanine in the crystal structure of the PP1{gamma}1/GM complex was sufficiently large to accommodate also a tryptophan side chain without requiring side chain rearrangement (Fig. 9E). Incorporation of tryptophan also did not require a displacement of the backbone or the hydrogen bonds between residues of the modeled motifs and Leu-289 and Cys-291 in PP1{gamma}1.

The threonine at position +4 exhibited a highly solvent-exposed side chain and was part of a hydrogen-bonding network in which a water molecule bridges its side chain hydroxyl and the backbone carbonyl of residue +2 to the main-chain carbonyl group of Thr-288 of PP1{gamma}1 (Fig. 9F). The same interaction was reported for the side chain hydroxyl group of a serine at the corresponding position in the PP1{gamma}1/GM complex (Fig. 9F) but was disrupted upon introducing an alanine (Fig. 6). The residue at position +6 is also solvent-exposed but did not form specific side chain interactions, consistent with the sequence variability observed at this position and its possible deletion without affecting binding behavior (Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Metabotropic glutamate receptors are dynamically regulated by associated proteins that are grouped together in macromolecular signaling complexes. The intracellular C termini of mGluRs define specific receptor isoforms and are prominent targets for interacting proteins. Here, we identified the C termini of the group I mGluRs, mGluR1a, and mGluR5a/b, as binding partners of the PP1C isoforms PP1{gamma}1/2. In an earlier study we showed that mGluR7b, a member of the group III mGluRs, also interacts with PP1{gamma}1/2 (31). Thus, in contrast to the specific binding between type I mGluRs and proteins of the Homer family (19, 22), the interaction between mGluRs and PP1{gamma}1/2 is not restricted to a specific receptor group.

We demonstrated co-expression of PP1{gamma}1/2 and interacting mGluRs in several regions of the mammalian CNS but could find nearly no overlapping distribution in organs outside the CNS. The observed expression patterns of type I mGluRs are consistent with published data, showing that mGluR5a is predominantly expressed in the olfactory bulb, whereas in cortex and hippocampus mGluR5b is the main isoform (20). In the retina, we found PP1{gamma}1 in dendrites of rod bipolar cells post-synaptic to rod photoreceptor terminals. Because mGluR1a and mGluR5a/b are expressed in rod bipolar cell dendrites (39), this co-localization of PP1{gamma}1 and group I mGluRs in the same subcellular compartment suggests their interaction in vivo. PP1{gamma}1 and associated mGluRs are also co-localized in the same subcellular compartments of neurons in other brain areas, e.g. in hippocampal interneurons of the stratum oriens and in dendrites of cerebellar purkinje cells (7, 42–45).

Of the eight known mGluR types, mGluR1a, mGluR5a/b, and mGluR7b were identified as PP1{gamma}1/2 binding partners in this and in a previous study (31). We mapped amino acids within the mGluR C termini mediating the protein interactions and uncovered an identical architecture of the PP1{gamma}1/2 binding domains, indicating that the protein interactions occur via similar molecular mechanisms. This hypothesis is supported by the finding that identical domains of PP1{gamma}1/2 bound to the mGluR C termini. Finally, we modeled the peptide sequences of the identified PP1{gamma}1 binding motifs of mGluR1a (KSVSW) and of mGluR5a/b and mGluR7b (KSVTW) in a complex with the enzyme, based on the reported crystal structure of the PP1{gamma}1/GM complex (34). The obtained three-dimensional representations favors an extended backbone conformation forcing the side chains of adjacent residues alternatively pointing to the solvent and to the surface of PP1{gamma}1, which is consistent with our experimental data.

As reported in a previous study, an alanine scan covering the isoform-specific distal region of mGluR7b revealed no contribution of amino acids outside its PP1{gamma}1 binding motif KSVTW (46), consistent with our observation that the sequences KSVSW and KSVTW were both necessary and sufficient to interact with PP1{gamma}1. Thus, the identified sequences KSVSW and KSVTW seem to be the complete PP1{gamma}1 binding motifs in mGluR C termini. These motifs share common characteristics with an already discussed PP1C binding motif (R/K)(V/I)XF (X = any amino acid) found in many proteins interacting with the enzyme (34).

Interestingly, the recently identified PP1C nuclear-binding protein p99 contains the same sequence motif as present in mGluR5a/b and mGluR7b C termini (47). The authors provided evidence for a physical interaction between PP1{gamma}1 and a 16-amino acid peptide containing the sequence KSVTW. Exchanging its tryptophan by alanine prevented binding of the peptide to PP1{gamma}1. This is in agreement with our study, where mutation of tryptophan residues in PP1{gamma}1/2 binding motifs disrupted the protein interactions. In contrast, changing the same tryptophan into phenylalanine did not influence the interaction with PP1{gamma}1 (47), indicating that an aromatic system might be needed in the last position of the binding motif. Based on their data, the authors proposed a PP1C binding motif consisting of the sequence RKSVTW (47). To us it seems unlikely that the N-terminal arginine would be needed to bind PP1{gamma}1, because the corresponding amino acid positions in mGluR1a and mGluR5a/b contain a glycine and a glutamine in mGluR7b. Furthermore, deletion of these residues did not influence binding to PP1{gamma}1 significantly.

PKC phosphorylation of mGluRs interacting with PP1{gamma}1/2 was demonstrated (48) and caused desensitization of mGluR1a and mGluR5 isoforms (49, 50). PKC phosphorylated Ser-881 in mGluR5, located in the PP1{gamma}1/2 binding motifs identified in this study. PKC was also linked to mGluR7b via PICK1 (27), and PKC phosphorylation of the proximal C-terminal domain, identical between mGluR7a and mGluR7b, was shown (51).

In summary, our results provide a molecular description of the PP1{gamma}1/2 binding sites on interacting mGluRs and identify the new PP1C binding motifs KSVSW and KSVTW. Because mGluR-mediated signal transduction depends largely on its association with regulatory proteins such as kinases and phosphatases, this study provides a molecular basis for the understanding of metabotropic glutamatergic signaling.


   FOOTNOTES
 
* This work was supported in part by the Deutsche Forschungsgemeinschaft and a Heisenberg Fellowship (to J. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 49-9131-852-6205; Fax: 49-9131-852-2485; E-mail: ralf.enz{at}biochem.unierlangen.de.

1 The abbreviations used are: mGluR, metabotropic glutamate receptor; PP1, protein phosphatase 1; GST, glutathione S-transferase; PKC, protein kinase C; CNS, central nervous system; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; nt, nucleotide(s); Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank Paul Greengard (Rockefeller University, New York, NY) for PP1{gamma}1 antibodies, Jeremy Nathans (The Johns Hopkins University, Baltimore, MD) for yeast two-hybrid plasmids, Erika Jung-Körner, Ines Walter, Nadja Schröder, and Anja Hildebrand for excellent technical assistance, Cord-Michael Becker and Adaling Ogilvie for helpful discussions, and Hans-Georg Breitinger and Stefan Stamm for critically reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schoepp, D. D. (2001) J. Pharmacol. Exp. Ther. 299, 12-20[Abstract/Free Full Text]
  2. Conn, P. J., and Pin, J. P. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 205-237[CrossRef][Medline] [Order article via Infotrieve]
  3. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., and Nakanishi, S. (1991) Nature 349, 760-765[CrossRef][Medline] [Order article via Infotrieve]
  4. Abe, T., Sugihara, H., Nawa, H., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1992) J. Biol. Chem. 267, 13361-13368[Abstract/Free Full Text]
  5. Baude, A., Nusser, Z., Roberts, J. D. B., Mulvihill, E., McIlhinney, R. A. J., and Somogyi, P. (1993) Neuron 11, 771-787[CrossRef][Medline] [Order article via Infotrieve]
  6. Nusser, Z., Mulvihill, E., Streit, P., and Somogyi, P. (1994) Neuroscience 61, 421-427[CrossRef][Medline] [Order article via Infotrieve]
  7. Lujan, R., Roberts, J. D., Shigemoto, R., Ohishi, H., and Somogyi, P. (1997) J. Chem. Neuroanat. 13, 219-241[CrossRef][Medline] [Order article via Infotrieve]
  8. Tanabe, Y., Nomura, A., Masu, M., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1993) J. Neurosci. 13, 1372-1378[Abstract]
  9. Ohishi, H., Ogawa-Meguro, R., Shigemoto, R., Kaneko, T., Nakanishi, S., and Mizuno, N. (1994) Neuron 13, 55-66[CrossRef][Medline] [Order article via Infotrieve]
  10. Shigemoto, R., Kinoshita, A., Wada, E., Nomura, S., Ohishi, H., Takada, M., Flor, P. J., Neki, A., Abe, T., Nakanishi, S., and Mizuno, N. (1997) J. Neurosci. 17, 7503-7522[Abstract/Free Full Text]
  11. Nakajima, Y., Iwakabe, H., Akazawa, C., Nawa, H., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1993) J. Biol. Chem. 268, 11868-11873[Abstract/Free Full Text]
  12. Okamoto, N., Hori, S., Akazawa, C., Hayashi, Y., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1994) J. Biol. Chem. 269, 1231-1236[Abstract/Free Full Text]
  13. Duvoisin, R. M., Zhang, C., and Ramonell, K. (1995) J. Neurosci. 15, 3075-3083[Abstract]
  14. Brandstätter, J. H., Koulen, P., Kuhn, R., van der Putten, H., and Wässle, H. (1996) J. Neurosci. 16, 4749-4756[Abstract/Free Full Text]
  15. Shigemoto, R., Kulik, A., Roberts, J. D., Ohishi, H., Nusser, Z., Kaneko, T., and Somogyi, P. (1996) Nature 381, 523-525[CrossRef][Medline] [Order article via Infotrieve]
  16. Takahashi, T., Forsythe, I. D., Tsujimoto, T., Barnes-Davies, M., and Onodera, K. (1996) Science 274, 594-597[Abstract/Free Full Text]
  17. Masu, M., Iwakabe, H., Tagawa, Y., Miyoshi, T., Yamashita, M., Fukuda, Y., Sasaki, H., Hiroi, K., Nakamura, Y., Shigemoto, R., Takada, M., Nakamura, K., Nakao, K., Katsuki, M., and Nakanishi, S. (1995) Cell 80, 757-765[CrossRef][Medline] [Order article via Infotrieve]
  18. Vardi, N., Duvoisin, R., Wu, G., and Sterling, P. (2000) J. Comp. Neurol. 423, 402-412[CrossRef][Medline] [Order article via Infotrieve]
  19. Brakeman, P. R., Lanahan, A. A., O'Brien, R., Roche, K., Barnes, C. A., Huganir, R. L., and Worley, P. F. (1997) Nature 386, 284-288[CrossRef][Medline] [Order article via Infotrieve]
  20. Minakami, R., Jinnai, N., and Sugiyama, H. (1997) J. Biol. Chem. 272, 20291-20298[Abstract/Free Full Text]
  21. Ishikawa, K., Nash, S. R., Nishimune, A., Neki, A., Kaneko, S., and Nakanishi, S. (1999) Genes Cells 4, 381-390[Abstract]
  22. Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan, A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron 23, 583-592[CrossRef][Medline] [Order article via Infotrieve]
  23. Ciruela, F., and McIlhinney, R. A. (2001) J. Neurochem. 76, 750-757[CrossRef][Medline] [Order article via Infotrieve]
  24. Ciruela, F., Escriche, M., Burgueno, J., Angulo, E., Casado, V., Soloviev, M. M., Canela, E. I., Mallol, J., Chan, W. Y., Lluis, C., McIlhinney, R. A., and Franco, R. (2001) J. Biol. Chem. 276, 18345-18351[Abstract/Free Full Text]
  25. Kitano, J., Kimura, K., Yamazaki, Y., Soda, T., Shigemoto, R., Nakajima, Y., and Nakanishi, S. (2002) J. Neurosci. 22, 1280-1289[Abstract/Free Full Text]
  26. O'Connor, V., El Far, O., Bofil-Cardona, E., Nanoff, C., Freissmuth, M., Karschin, A., Airas, J. M., Betz, H., and Boehm, S. (1999) Science 286, 1180-1184[Abstract/Free Full Text]
  27. El Far, O., Airas, J., Wischmeyer, E., Nehring, R. B., Karschin, A., and Betz, H. (2000) Eur. J. Neurosci. 12, 4215-4221[CrossRef][Medline] [Order article via Infotrieve]
  28. Enz, R. (2002) FEBS Lett. 514, 184-188[CrossRef][Medline] [Order article via Infotrieve]
  29. Hirbec, H., Perestenko, O., Nishimune, A., Meyer, G., Nakanishi, S., Henley, J. M., and Dev, K. K. (2002) J. Biol. Chem. 277, 15221-15224[Abstract/Free Full Text]
  30. Saugstad, J. A., Yang, S., Pohl, J., Hall, R. A., and Conn, P. J. (2002) J. Neurochem. 80, 980-988[CrossRef][Medline] [Order article via Infotrieve]
  31. Enz, R. (2002) J. Neurochem. 81, 1130-1140[CrossRef][Medline] [Order article via Infotrieve]
  32. Croci, C., Brandstätter, J. H., and Enz, R. (2003) J. Biol. Chem. 21, 6128-6135
  33. da Cruz e Silva, E. F., Fox, C. A., Ouimet, C. C., Gustafson, E., Watson, S. J., and Greengard, P. (1995) J. Neurosci. 15, 3375-3389[Abstract]
  34. Egloff, M. P., Johnson, D. F., Moorhead, G., Cohen, P. T., Cohen, P., and Barford, D. (1997) EMBO J. 16, 1876-1887[CrossRef][Medline] [Order article via Infotrieve]
  35. Powell, M. J. D. (1977) Mathemat. Progr. 12, 241-254[CrossRef]
  36. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr., 26, 283-291[CrossRef]
  37. Hooft, R. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve]
  38. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Protein Eng. 8, 127-134[Abstract/Free Full Text]
  39. Koulen, P., Kuhn, R., Wässle, H., and Brandstätter, J. H. (1997) J. Neurosci. 17, 2200-2211[Abstract/Free Full Text]
  40. Connor, J. H., Kleeman, T., Barik, S., Honkanen, R. E., and Shenolikar, S. (1999) J. Biol. Chem. 274, 22366-22372[Abstract/Free Full Text]
  41. Zhang, Z., Zhao, S., Bai, G., and Lee, E. Y. (1994) J. Biol. Chem. 269, 13766-13770[Abstract/Free Full Text]
  42. Petralia, R. S., Wang, Y. X., Singh, S., Wu, C., Shi, L., Wei, J., and Wenthold, R. J. (1997) J. Chem. Neuroanat. 13, 77-93[CrossRef][Medline] [Order article via Infotrieve]
  43. Kinoshita, A., Shigemoto, R., Ohishi, H., van der Putten, H., and Mizuno, N. (1998) J. Comp. Neurol. 393, 332-352[CrossRef][Medline] [Order article via Infotrieve]
  44. Strack, S., Kini, S., Ebner, F. F., Wadzinski, B. E., and Colbran, R. J. (1999) J. Comp. Neurol. 413, 373-384[CrossRef]