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Originally published In Press as doi:10.1074/jbc.M307120200 on August 11, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42551-42559, October 24, 2003
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Molecular Similarities in the Ligand Binding Pockets of an Odorant Receptor and the Metabotropic Glutamate Receptors*

Donghui Kuang{ddagger}, Yi Yao{ddagger}, Minghua Wang{ddagger}, N. Pattabiraman§, Lakshmi P. Kotra{ddagger}, and David R. Hampson{ddagger}

From the {ddagger}Department of Pharmaceutical Sciences and Institute for Drug Research, University of Toronto, Toronto, Ontario M5S 2S2, Canada and the §Department of Oncology Lombardi Cancer Center, Georgetown University, Washington D. C. 20057

Received for publication, July 3, 2003 , and in revised form, August 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the {alpha} amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Of the estimated 800-950 G-protein-coupled receptor genes in the human genome, approximately one-half are olfactory or taste receptor genes, whereas the remainder encode receptors for endogenous ligands (1-3). Although many olfactory and putative pheromone receptors have been cloned or identified through genome sequencing efforts, the elucidation of the ligands or odorants that activate individual olfactory receptors has proven to be a difficult task. One of the relatively few examples of a match between a cloned receptor and an odorant is the "5.24 receptor." This receptor is expressed in the sensory epithelium of fish and is activated by several amino acids, the most potent of which are the basic amino acids L-lysine and L-arginine (4). The 5.24 receptor belongs to the family 3 subclass of G-protein-coupled receptors and shares amino acid sequence similarity with the V2R class of mammalian pheromone receptors (5-7), the calcium-sensing receptor (CaR1 (8-10)), mammalian taste receptors (11, 12), and the metabotropic glutamate receptors (mGluRs (13)). Based in part on the observation that about 40% of the neurons in the catfish olfactory epithelium respond to L-arginine (14), it appears likely that the 5.24 receptor plays a role in navigation and feeding in fish. In teleost and elasmobranch fish, the homologous CaR operates as an ion sensor for probing changes in the salinity of the surrounding water (15).

The 5.24 odorant receptor shares 26-28% amino acid sequence identity with the rat mGluRs and 32% identity with the rat CaR. Notably, most of the highly conserved cysteines present in all eight members of the mGluR family and the homologous CaR are also conserved in the 5.24 lysine receptor. Several of the cysteines in the mGluRs and the CaR form intramolecular disulfide bridges within a receptor monomer, and at least one forms an intermolecular disulfide bridge linking two monomers (16-19). The glutamate binding pocket in the mGluRs is contained within the large extracellular amino-terminal domain of the protein (20-22). Although earlier molecular models (23-25) were based on the x-ray structures of the periplasmic-binding proteins from bacteria, which have low sequence homology to the mGluRs, the current homology models are based on the crystal structure of the rat mGluR1 receptor (19).

The objective of the present study was to explore the molecular architecture within the proposed amino acid binding pocket of the 5.24 receptor. We sought to identify differences between the 5.24 receptor and the various subtypes of mGluRs in both the conserved and non-conserved residues that interact with the {alpha} amino and {alpha} carboxyl groups of the ligand. Of particular interest was the identification of residues that establish bonding interactions with the side chain of the bound lysine ligand. The results obtained shed light on the molecular determinants of amino acid selectivity and support the concept of a highly conserved amino acid recognition motif in family 3 G-protein-coupled receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—L-Lysine and L-arginine were from Aldrich Chemical Co. L-[4,5-3H(N)]Lysine (specific activity 98.5 Ci/mmol) was purchased from PerkinElmer Life Sciences, Inc., Canada. Oligonucleotides were synthesized by the Mobix Laboratory (McMaster University, Hamilton, Canada). The anti-c-myc mouse monoclonal antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). The peroxidase-labeled anti-mouse IgG secondary antibody was from Amersham Biosciences Canada (Oakville, Ontario, Canada).

Expression Constructs and Site-directed Mutagenesis—The goldfish 5.24 odorant receptor cDNA in the human cytomegalovirus-1 vector was kindly provided by Dr. John Ngai (University of California, Berkeley, CA). To subclone the full-length wild-type 5.24 cDNA into the mammalian expression vector pcDNA3.1 A c-myc-His (Invitrogen), a three-piece ligation was performed. The first piece was a 2.5-kb fragment yielded by a restriction digestion of the full-length 5.24 cDNA in CMVI with EcoRI and BstXI. The second piece was from a PCR conducted on the full-length 5.24 cDNA in CMVI template using a forward primer (5'-CTTGCACAGGCCTTTTATTG-3') and a reverse primer (5'-GCTCTAGAAAGCACATTGTCTATTTCTGG-3'). The forward primer contained a BstXI restriction site. The reverse primer included an XbaI site to delete the stop codon and 3'-untranslated region and incorporate the nucleotide sequence encoding the c-myc and polyhistidine (His) epitope tags. The PCR product was digested with BstXI and XbaI to generate a 497-bp fragment. The 497-bp BstXI-XbaI PCR fragment and the 2.5-kb EcoRI-BstXI 5.24 fragment were then ligated into the EcoRI-XbaI fragment of pcDNA3.1 A c-myc-His vector.

A three-piece ligation was also used to generate the truncated 5.24 cDNA construct. The first piece was a 1.0-kb fragment generated by digestion of 5.24 cDNA in human cytomegalovirus-1 by EcoRI and AccI. The second piece was obtained from a PCR conducted on the full-length 5.24 cDNA in the pcDNA3.1 A c-myc-His template using a forward primer (5'-GAGGGCCTGCGTAGTAGGAACGTTCC-3') and a reverse primer (5'-GCTCTAGAATCTGTGTGGTTGGAGTATTG-3'). The forward primer contained an AccI restriction site, and the reverse primer included an XbaI site to truncate the amino-terminal domain of 5.24 receptor immediately after aspartate 533. The PCR product was digested with AccI and XbaI to generate a 669-bp fragment. The third fragment was produced from the modified pcDNA 3.1 A vector, which contained a deletion of the polyHis tag. Finally, the modified pcDNA 3.1 A c-myc vector was then ligated with the 669-bp AccI-XbaI 5.24 PCR fragment and the 1.0-kb EcoRI-AccI 5.24 fragment to obtain the construct of 5.24 ATD in pcDNA 3.1 A c-myc vector.

All the mutations in the mutants were made using the QuikChangeTM site-directed mutagenesis strategy (Stratagene). The 665-bp EcoRV cassette of 5.24 was subcloned into pBluescript KS+ (Stratagene) and used as a template for all of the mutants except K74A, Q78A, and D388A, which were mutated in a 1.6-kb EcoRI-XbaI cassette in pKS+. Two complementary oligonucleotides (25-45 bases) containing the desired base pair changes in the middle with ~10-15 bases of the same sequences as the wild-type on both sides were used as primers for each mutation. PCRs were performed using Pfu Turbo DNA polymerase (Stratagene) for 16 cycles in a programmable thermal controller (MJ Research Inc.). DpnI was added to each amplification reaction to digest the parental DNA template. The mutated double-stranded DNAs were transformed into Escherichia coli DH5 (Invitrogen)-competent cells, purified and sequenced for confirmation of mutations. The mutated cassettes were then subcloned back into the truncated or full-length wild-type 5.24 constructs. The integrity of all DNA constructs and mutants were confirmed by automated DNA sequencing using a LiCor LongReadIR automated sequencer.

Plasmid Preparations and Transient Transfections—cDNA constructs were transformed into E. coli DH5{alpha}-competent cells. Ampicillin-resistant colonies were inoculated into 3 ml of Luria-Bertani medium containing ampicillin and grown at 37 °C for 16 h. The plasmid DNAs for transfections were prepared using QIAspin Miniprep kit (Qiagen). Plasmid DNAs were transiently transfected into HEK-293 or HEK-293 TSA-201 cells by the calcium phosphate methods as described previously (21) or in the functional assays using the FuGENE 6 reagent (Roche Applied Science) or LipofectAMINE 2000 (Invitrogen) as described by the manufacturers. The cells were cultured in minimal essential medium (Invitrogen) with 6% fetal bovine serum (HyClone, Inc.) and antibiotics. For the truncated secreted receptors, 24 h after transfection, the cell culture medium was replaced with Opti-MEM (Invitrogen). Forty-eight h after transfection, the culture media containing the secreted truncated receptors and the remaining whole cells attached to the cell culture plates (i.e. the total cell fraction for the full-length receptor) were collected separately and processed as described below.

Soluble Protein Preparation and Radioligand Binding Assays—The cell culture medium was collected and centrifuged at 48,400 x g for 15 min at 4 °C, and the supernatant was dialyzed at 4 °C in 800 ml of binding buffer (20 mM HEPES, 1 mM EDTA, 1 mM MgCl2·6H2O, pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride and 1 mM EGTA; the dialysis buffer was changed three times over a 24-h period. The samples were pooled, centrifuged at 48,400 x g for 45 min at 4 °C, and stored at -70 °C. For radioligand binding, the samples were thawed and re-centrifuged at 48,400 x g for 45 min at 4 °C. 200 µl of soluble protein was used for all binding assays in a final volume of 250 µl. All binding assays were performed on ice using 50 nM [L-3H]lysine; nonspecific binding was defined as binding in the presence of 500 µM L-arginine. Competition assays were performed with 50 nM [L-3H]lysine and increasing concentrations of L-lysine. Following a 40-min incubation, 340 µg of {gamma}-globulin and 200 µl of 30% cold polyethylene glycol were added to each sample. The mixture was incubated on ice for 4 min and centrifuged at 13,200 x g for 4 min. The supernatant fraction was aspirated. The pellet was washed with 500 µl of cold 15% polyethylene glycol and solubilized in 500 µl of 1 M NaOH for overnight. The retained radioactivity was measured by scintillation counting. The data were analyzed using GraphPad Prism 3.0 (GraphPad Software, Inc.).

Deglycosylation—For membrane proteins, 20 µg of protein was suspended in 18 µl of phosphate-buffered saline. After added 2 µl of 10x denaturing buffer (New England Biolabs, Beverley, MA), the samples were incubated at 37 °C for 15 min. Subsequently, 3 µl of 10x G7 Buffer (New England Biolabs), 3 µl of 10% Nonidet P-40 (New England Biolabs), and 4 µl of PNGase F (New England Biolabs) was added. After a 1-h incubation at 37 °C, 4x SDS sample buffer with 100 mM dithiothreitol (final concentration) was added, and the samples were subjected to electrophoresis and immunoblotting. For deglycosylation of the secreted receptor, 200 µl of dialyzed sample of soluble receptor was concentrated to 100 µl, and 18 µl of the condensed sample was subjected to the same procedure described above.

Immunoblotting—SDS-PAGE and immunoblotting with the anti-c-myc mouse monoclonal antibody were conducted as described previously (21). 5 µg of total cellular protein was loaded for analysis of membrane proteins. For the analysis of secreted proteins, media from transfected HEK cells were collected, dialyzed in binding buffer, and concentrated about 7-fold prior to electrophoresis and immunoblotting. For treatment with the proteasome inhibitor Z-Leu-Leu-Leu-aldehyde (MG-132), 24 h post-transfection the minimum essential medium was replaced by Opti-MEM medium supplemented with 10 µM MG-132. Six hours later, another aliquot of 10 µM MG-132 was added into the medium. Forty-eight hours after the transfection, the culture medium and the cells were collected and subjected to SDS-PAGE.

Functional Analysis of Wild-type and Mutant Receptors—Twenty-four hours after transfection, HEK cells were plated in polyornithine pre-treated 96-well microtiter plates (Costar black microtiter plates) at 100,000 cells/100 µl of minimal essential medium. Forty hours post-transfection, cells were washed once with calcium assay buffer (20 mM HEPES, pH 7.4, 146 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml bovine serum albumin, and 1 mg/ml glucose) and incubated at 37 °C in 100 µl of assay buffer. After a 2-h incubation, the assay buffer was changed and the cells were incubated for another 2 h at 37 °C. The assay buffer was removed, and 30 µl of assay buffer containing 6 µM of Fluo-4 (Molecular Probes Inc.) was added into each well; the cells were incubated for 1 h at room temperature in the dark. The cells were washed three times with assay buffer and then incubated in 150 µl of assay buffer for 30 min at room temperature in the dark. L-Lysine dissolved in assay buffer was added, and the responses were recorded on a FLEXstation benchtop scanning fluorometer (Molecular Devices Corp., Sunnyvale, CA) at room temperature with settings of 485 nm for excitation and 525 nm for emission. GraphPad Prism 3.0. software was used to plot fluorescence intensities and calculate the EC50 values.

Molecular Modeling—A homology model of the closed form of the extracellular domain of the fish 5.24 was generated using the x-ray crystal structure of the extracellular domain of rat mGluR1 as a template (Ref. 19; PDB coordinates, 1EWK [PDB] ). The MODELER version 6 program (26) was used to generate the homology model, and Sybyl 6.9 (Tripos Inc., St. Louis, MO) was used to view, analyze, and manipulate the structure. The sequence alignment between the rat mGluR1 and fish 5.24 sequences was obtained from a multiple sequence alignment that included the eight subtypes of mGluRs, the CaR, and the fish 5.24 (Fig. 1). The alignment was then modified to preserve the conserved cysteines and to avoid unreasonable gap insertions in conserved secondary structure motifs. A few residues located in a loop structure of the 5.24 receptor and outside the binding pocket were manually adjusted to be consistent with the secondary structure of the protein in the mGluR1 x-ray structure. The fish 5.24 homology model has no intramolecular short steric contacts, and the backbone torsion angles are within the allowed regions in the Ramachandran phi-psi plot (27). The coordinates for the zwitterionic form of lysine were generated in an extended conformation using Sybyl 6.9. L-Lysine was manually docked into the binding pocket; the orientation of the {alpha} amino and carboxyl groups of the docked lysine ligand was the same as that of the bound glutamate in the mGluR1 structure. The complex was solvated in a water box (three point transferable intermolecular potentials) so that the box boundaries are at least 10 Å away from any protein surface atoms. The coordinates for the solvated complex were then energy-minimized using AMBER 7.0 (28) and the Cornell force field (29). The energy minimization was carried out until the maximum derivative was less than 0.001 kcal/mol/Å.



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  		FIG. 1.
Multiple sequence alignment of a portion of the amino-terminal domains of the rat mGluRs, the rat calcium-sensing receptor (CaR), and the goldfish 5.24 odorant receptor. The alignment was made using ClustalW 1.81. All sequences begin from the first conserved glycine just downstream from the putative signal peptide cleavage sites. Asterisks at the top denote residues in the binding pocket of the mGluR1 crystal structure. Amino acids mutated in the 5.24 sequence are highlighted with a black background.

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To facilitate the monitoring of protein expression on immunoblots of transfected HEK cells, the 5.24 receptor cDNA was subcloned into the pcDNA3.1 expression vector to produce a fusion protein with a c-myc epitope tag incorporated into the carboxyl terminus. Initial experiments conducted on this expression construct indicated that the protein was expressed as determined via immunoblotting with a c-myc antibody. However, preliminary radioligand binding experiments carried out using either [L-3H]lysine or [L-3H]arginine showed a high level of binding with both ligands in membranes from mock-transfected HEK cells not expressing the 5.24 receptor (not shown). Attempts to reduce this background binding were only partially successful.

As an alternative strategy, [L-3H]lysine or [L-3H]arginine binding was examined on a truncated version of the 5.24 receptor that encompassed most of the extracellular domain of the protein. A stop codon was mutated into this cDNA construct after the c-myc tag to eliminate the polyHis portion of the fusion protein; this later alteration facilitated protein expression and secretion from transfected cells. The site of truncation in the 5.24 receptor (aspartate 533) was upstream from the first putative transmembrane domain and the cysteine-rich region of the molecule and was similar to the site of truncation previously made in the mGluRs in which ligand binding was retained (21, 22). As with the truncated mGluRs, the truncated 5.24 lysine receptor was secreted from transfected HEK cells into the cell culture medium.

Both monomeric and dimeric forms of the 5.24 of the membrane-bound full-length receptor were visible on immunoblots of transfected HEK cells. Only the monomeric form of the truncated receptor was detected under reducing conditions. The monomeric forms of the full-length membrane-bound receptor and the truncated 5.24 lysine receptor migrated on SDS-PAGE at relative molecular weights of 143,000 and 109,000 (Fig. 2A, lanes 2 and 4, respectively); in both cases the molecular masses of the proteins were about 40-50 kDa larger than the predicted masses of (98,131 and 59,264 Da, respectively) based on amino acid sequence. Treatment of the full-length membrane-bound receptor and the secreted truncated form of the receptor with PNGase F, an enzyme that cleaves asparagine-linked oligosaccharides, resulted in both cases in shifts to a lower Mr (102,000 and 70,000; Fig. 2A, lanes 3 and 5, respectively).



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  		FIG. 2.
Enzymatic deglycosylation of the full-length membrane-bound and the truncated soluble ligand binding domain of the 5.24 lysine receptor. Whole cell fractions or cell culture medium from transfected HEK cells were separated on 8% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with an anti-c-myc monoclonal antibody. Panel A: lane 1, whole cell fraction from mock-transfected cells; lane 2, whole cell fraction from cells expressing the full-length 5.24 receptor; lane 3, whole cell fraction from 5.24-expressing cells treated with the deglycosylating enzyme PNGase F; lane 4, cell culture medium from HEK cells transfected with truncated 5.24 receptor; lane 5, cell culture medium from cells transfected with truncated lysine receptor and treated with PNGase F; lane 6, cell culture medium from mock-transfected cells. Panel B: effects of enzymatic deglycosylation on [L-3H]lysine binding to the truncated 5.24 receptor.

 

The observation that the truncated and full-length receptors did not run at the predicted molecular weights after deglycosylation suggests that other post-translational modifications may be present on the molecule. However, the deglycosylation experiments demonstrated that both the intact membrane-bound receptor and the secreted 5.24 receptor are highly glycosylated, a finding that is consistent with the presence of 14 potential consensus sequences for asparagine-linked carbohydrates in the extracellular domain of the protein. Moreover, enzymatic deglycosylation of the secreted truncated 5.24 receptor did not reduce the binding of [L-3H]lysine compared with the untreated receptor (Fig. 2B).

To conduct radioligand binding experiments on the truncated secreted form of the 5.24 lysine receptor, the media overlaying the transfected cells was collected 48 h after transfection, dialyzed, and then analyzed in a modified microcentrifugation radioligand binding assay originally adopted for use on soluble secreted forms of the mGluRs (21, 22). Initial binding experiments on media collected from mock-transfected HEK cells also indicated the presence of some background binding with both [L-3H]lysine or [L-3H]arginine, albeit much lower than was seen with the full-length receptor. However, we found that freezing samples of the truncated receptor preparation prior to conducting the binding assay alleviated the interfering background binding while having minimum impact on the binding of [L-3H]lysine or [L-3H]arginine to the 5.24 receptor (see Fig. 4A below). Thus in all subsequent binding studies, the secreted receptor preparations were frozen and thawed before analysis.



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  		FIG. 4.
Protein expression and [L-3H]lysine binding to mutants of the truncated 5.24 receptor. Equivalent residues in the rat mGluRs, and the rat calcium-sensing receptor are shown in Table II. The bands above the histogram indicate the level of protein expression on immunoblots using an anti-c-myc mouse monoclonal antibody. The histograms indicate the relative levels of specific [L-3H]lysine binding (at 50 nM) in each of the mutant receptors compared with the wild-type 5.24 receptor. The data shown are the means ± S.E. of three to four experiments. The mutants are grouped according to the proposed interactions with the functional groups on the bound lysine ligand. A, mutants interacting with the {alpha} carboxyl group; B, mutants interacting with the {alpha} amino group; C, mutants interacting with the terminal amino group on the side chain of the bound lysine ligand; D, proteasome-regulated degradation of the truncated T175A mutant. Twenty-four hours after transfection, HEK cells were treated for an additional 24 h with 10 µM of the proteasome inhibitor MG-132; samples were prepared for SDS-PAGE and probed with an anti-c-myc monoclonal antibody. Lane 1, whole cell sample from mock-transfected cells; lane 2, whole cell sample from cells transfected with T175A truncated mutant and treated with MG-132; lane 3, whole cell sample from cells transfected with T175A truncated mutant without treatment with MG-132. The arrowhead indicates the position of the truncated 5.24 receptor in lane 2.

 


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  		TABLE II
Amino acid conversion chart for residues mutated in the 5.24 lysine receptor

Conversion chart for residues mutated in the 5.24 lysine receptor. Residues labeled with an asterisk are present in the glutamate binding pocket of the mGluR1 crystal structure. Mutations of amino acids highlighted with a grey background resulted in either elimination of ligand binding or a 10-fold or greater decrease in ligand affinity. Numbering for the CaR and the mGluRs refers to the rat sequences.

 
Under these conditions, in radioligand binding competition experiments using unlabeled lysine competing with [L-3H]lysine the inhibition constant was IC50 = 0.43 ± 0.19 µM (Fig. 3); this value is very similar to that reported previously with the full-length membrane-bound receptor using [L-3H]arginine (4). Inhibition experiments using L-arginine and L-ornithine also yielded inhibition constants (Fig. 3A) that were similar to those reported for the full-length receptor using [L-3H]arginine (4). D-Lysine was an extremely poor competitor of [L-3H]lysine binding with an IC50 value of 0.27 ± 0.02 mM, indicating that the stereospecific configuration surrounding the {alpha} carbon atoms in the lysine molecule is an important determinant for high affinity binding. The effects of the ionic components of the binding buffer were also examined. [L-3H]Lysine binding to the truncated 5.24 receptor was highly dependent on the presence of chloride ions but not on the presence of magnesium ions (Fig. 3B). The dependence of ligand binding of the 5.24 receptor on chloride ions is consistent with the chloride dependence of agonists to some subtypes of mGluRs (30).



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  		FIG. 3.
Pharmacological analysis of the truncated soluble 5.24 lysine receptor. A, radioligand binding competition experiments carried out using [L-3H]lysine (50 nM) and various concentrations of unlabeled L-lysine, D-lysine, L-arginine, and L-ornithine. The IC50 values (mean ± S.E.) calculated from the data were: L-lysine, 0.43 ± 0.19 µM; D-lysine, 0.27 ± 0.02 mM; L-arginine, 0.31 ± 0.30 µM; L-ornithine, 1.1 ± 0.36 µM. Each value is the average of two to four determinations conducted in triplicate. B, ion requirements for [L-3H]lysine binding to the 5.24 receptor. The normal (control) binding buffer consisted of HEPES 10 mM, EDTA 1.0 mM, and MgCl2·6H2O 1.0 mM (pH = 7.4). The HEPES plus EDTA buffer excluded MgCl2 (no chloride and no magnesium), whereas in the third condition, MgCl2 was replaced with MgSO4 (no chloride) (n = 3).

 

Using the molecular coordinates from the crystal structure of mGluR1 as the template, we constructed a computer-generated model of the 5.24 lysine receptor. L-Lysine was "docked" manually into a position analogous to the bound L-glutamate in the closed form of mGluR1. This model (see below) was used as a guide to explore the proposed ligand binding pocket via site-directed mutagenesis. Single point mutations were made in the truncated version of the 5.24 receptor by converting the natural amino acid to alanine, and in the case of threonine 175, to either alanine or serine. Protein expression and [L-3H]lysine binding of the mutants were then compared with the truncated unmutated receptor.

A summary of the radioligand and protein expression experiments is shown in Fig. 4. For each of the truncated mutants, which incorporated an c-myc epitope tag into the carboxyl terminus, the relative level of protein expression was monitored on immunoblots using an anti-c-myc monoclonal antibody. The mutants portrayed in Fig. 4 are arranged according to the projected interactions between amino acids in the binding pocket and the functional groups on the docked lysine ligand. Thus serines 151 and 152 and lysine 283 were projected to interact with the {alpha} carboxyl group of L-lysine (Fig. 4A), whereas the {alpha} amino group of the lysine ligand was predicted to establish bonds with threonine 175, aspartate 195, tyrosine 223, and aspartate 309 (Fig. 4B). Additional residues, including lysine 74, glutamine 78, serine 111, serine 150, arginine 190, serines 284 and 285, and aspartate 388 were postulated to potentially interact with the distal amino group on the side chain of L-lysine (Fig. 4C).

With the exception of the threonine 175 to alanine mutant (T175A), all of the truncated mutants generated were secreted from transfected HEK cells at levels similar to the truncated wild-type receptor. Unlike all the other mutants, including the full-length T175A mutant (see below), the truncated T175A mutant showed no protein expression on immunoblots. However, when threonine 175 was conservatively mutated to serine (T175S) instead of alanine, wild-type protein expression and [L-3H]lysine binding was observed. The absence of secreted protein for the truncated T175A mutant could be due to proteasome-mediated degradation of the receptor. To test this possibility, transfected cells were treated with the membrane-permeable proteasome inhibitor MG-132. In cells pretreated with MG132 the mutant protein was detected in whole cells but not in the overlaying medium (Fig. 4D). This observation suggests that conversion of threonine 175 to alanine caused mis-folding and trapping of the protein inside cells with subsequent rapid intracellular proteolytic degradation. The data depicted in Fig. 4B indicate that mutation to alanine of the other three amino acid side chains that interact with the {alpha} amino group of the bound lysine all had a dramatic impact on ligand binding. The D195A and Y223A mutations completely eliminated [L-3H]lysine binding, whereas the D309A mutation caused a 94% loss of binding activity.

Mutations in the three amino acids postulated to contact the {alpha} carboxylic acid group of the bound lysine ligand, serine 151, serine 152, and lysine 283 displayed 79, 55, and 1%, respectively, of the [L-3H]lysine binding observed in the wild-type receptor (Fig. 4A). The remaining amino acids, including Lys-74, Gln-78, Arg-190, Ser-111, Ser-150, Ser-284, Ser-285, and Asp-388, were postulated to potentially establish bonding interactions with the distal amino group in the side chain of the lysine ligand in the wild-type receptor. The K74A, S111A, S150A, S284A, and S285A mutants displayed levels of binding and protein expression that were very similar to the wild-type receptor. The Q78A, S152A, and R190A mutants showed reductions in binding assays using a single concentration of [L-3H]lysine (50 nM, Fig. 4C); in these instances sufficient specific binding was retained to conduct a more detailed analysis of the ligand binding properties. Therefore, autocompetition experiments using tritiated and unlabeled L-lysine were carried out. The IC50 values for the Q78A, S152A, and R190A mutants were 1.1 ± 0.16, 0.43 ± 0.26, and 0.94 ± 0.14 µM, respectively. Thus, these mutations produced either no change (S152A) or relatively minor decreases (Q78A, R190A) in affinity compared with the truncated wild-type receptor (Fig. 5). In contrast, mutation of aspartate 388, projected to establish an interaction with the side-chain amino group of L-lysine, resulted in an 88% decrease in binding relative to the wild-type receptor (Fig. 4C).



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  		FIG. 5.
Radioligand binding to mutant 5.24 receptors. Competition experiments were carried out on the S152A, Q78A, and R190A truncated mutants using 50 nM [L-3H]lysine. Each point represents the mean ± S.E. of three determinations conducted in triplicate on samples obtained from three different transfections. The IC50 values (mean ± S.E.) were S152A, 0.43 ± 0.26 µM; Q78A, 1.1 ± 0.16 µM; R190A, 0.94 ± 0.14 µM.

 

As noted above, we were not able to perform radioligand binding assays on the full-length membrane-bound receptors due to a high background binding of [L-3H]lysine binding in mock-transfected HEK cells. However, functional analysis of the full-length mutant receptors was possible using a fluorescence-based assay on live transfected HEK cells. Previous work indicated that the 5.24 receptor is coupled to the stimulation of phosphoinositide turnover (4). Therefore, transfected cells were loaded with the calcium-sensitive dye Fluo-4 and challenged with various concentrations of L-lysine, and fluorescence measurements were made in 96-well microtiter plates.

Rapid dose-dependent increases in fluorescence were observed in cells expressing the wild-type 5.24 receptor after exposure to L-lysine (Fig. 6A). No responses to lysine were seen in untransfected cells at concentrations up to 5 mM; at concentrations of L-lysine above 5 mM, a large sustained elevation in baseline fluorescence was seen precluding quantitation in the millimolar range. The EC50 for the wild-type receptor was 2.9 ± 0.3 µM (Fig. 6B and Table I). The S111A mutant had an EC50 value that was about 2-fold greater affinity than the wild-type receptor, the S284A and S285A mutants had affinities that were very similar to the wild-type, and the S150A showed an affinity that was about 2.4-fold lower than the wild-type receptor. Surprisingly and in contrast to the radioligand binding results, which showed little change in binding affinity, the S152A mutant displayed a 25-fold decrease in affinity in the calcium release assay. The most dramatic effects were seen with the four residues interacting with the {alpha}-amino group of the lysine ligand (Thr-175, Asp-195, Tyr-223, and Asp-309), and the K283A mutant, which is expected to establish interactions with the {alpha} carboxyl group of the ligand. The EC50 values of the T175A and Y223A mutants showed affinities that were 93- and 169-fold lower, respectively, compared with the wild-type receptor. Only small responses were seen with the D195A mutant at 1 and 5 mM but not at lower concentrations. No responses were observed with the K283A and D309A mutants at any concentration of L-lysine tested (up to 5 mM).



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  		FIG. 6.
Functional analysis of the wild-type mutant receptors in live HEK cells. Transfected cells were plated into 96-well microtiter plates and loaded with the calcium-sensitive dye, Fluo-4. The cells were exposed to various concentrations of L-lysine and responses were recorded on a FLEXstation fluorescence plate reader (Molecular Devices). A, example of fluorescence intensity over time in the wild-type receptor after exposure to various concentrations of L-lysine. B, activation curves for the wild-type and S111A, S150A, S284A, and S285A mutants. C, activation curves for the wild-type and S152A, Y223A, and T175A mutants. D, activation curves for wild-type and the D388A mutant using L-lysine and L-glutamine as agonists. The EC50 values for L-lysine activation are listed in Table I. The EC50 values for L-glutamine activation of the wild-type and D388A mutant were 8.9 ± 1.5 µM (n = 4) and 7.9 ± 2.0 µM (n = 6), respectively.

 

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  		TABLE I
EC50 values for L-lysine activation of HEK cells expressing wild-type and mutant 5.24 receptors

The values are means ± S.E. of three to eight experiments.

 

Because the D388A mutation was the only mutant among the amino acids predicted to interact with the side chain of the bound ligand, which showed a large reduction in binding, additional analyses were conducted in the functional assay using both L-lysine and the polar amino acid L-glutamine as agonists. The EC50 values for L-lysine at the wild-type and D388A mutant were 2.9 ± 0.35 µM (n = 4) and 75.7 ± 6.0 µM (n = 8), respectively (Fig. 6D); this difference in affinity was highly significant (p < 0.001, t test). In contrast, the EC50 values for L-glutamine activation of the wild-type (8.9 ± 1.5 µM, n = 4) and the D388A mutant (7.9 ± 2.0 µM, n = 6) were similar and not significantly different (p = 0.719).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The primary objective of this study was to explore the molecular architecture within the proposed amino acid binding pocket of the 5.24 odorant receptor and to compare the mutagenesis results with those obtained in recent studies on the mGluRs and the CaR. The CaR, like the 5.24 receptor but unlike the mGluRs, possess an amino acid binding site that is broadly tuned to several amino acids (31). It was anticipated that at least some of the molecular determinants mediating bonds between the {alpha} amino and {alpha} carboxyl groups of the bound glutamate ligand in the mGluRs would also be important for L-lysine binding to the 5.24 receptor. It is important to note, however, that some of the amino acids mediating interactions with the {alpha} amino and {alpha} carboxyl group of the glutamate ligand are not conserved within the mGluR subtypes, (e.g. Ser-164, Glu-292, and Gly-293 in mGluR1 (Table II)). Moreover, with the residues that are conserved, the relative importance of each varies between the different subtypes of mGluR and the ligand used to activate (or block) the receptor (32).

A series of mutations was made in amino acids in the 5.24 receptor that were postulated to potentially establish favorable contacts with the terminal amino group on the side chain of the lysine ligand. The residues in this category were identified or predicted based on one or more of the following criteria: (a) were present within 6 Å of the bound lysine in the 5.24 receptor homology model (lysine 74, glutamine 78, serine 150, and aspartate 388); (b) aligned with a residue in the crystal structure of mGluR1 (serine 111 and serine 284; see Table II); or (c) aligned with an amino acid previously shown to be important for agonist activation in the mGluRs and/or the CaR (arginine 190).

Within this set of mutations, only the D388A mutant produced a large decrease in [L-3H]lysine binding and in ligand affinity in the calcium release assay. In our homology model, Asp-388 occupies a position equivalent to Lys-409 in mGluR1 that interacts with the terminal carboxyl group on the glutamate ligand. In the 5.24 receptor model, the oxygen atoms on the terminal carboxyl group of aspartate 388 are situated at 2.7-2.8 Å from the terminal amino group on the side chain of the docked lysine ligand (Fig. 7), suggesting that they form ionic or hydrogen bonds. In addition to L-lysine and L-arginine, the 5.24 odorant receptor is also activated by higher concentrations of several other amino acids, including L-glutamine (4). The lack of effect of the D388A mutation on the ability of L-glutamine to activate the receptor is compatible with our molecular model, because the shorter length of the L-glutamine side chain likely precludes a viable interaction with this residue in the binding pocket.



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  		FIG. 7.
The ligand binding site in the energy-minimized homology model of the 5.24 odorant receptor. Selected key amino acids are depicted in the proposed binding pocket for L-lysine. The docked lysine ligand is represented as a "ball-and-stick" model. The yellow lines depict hydrogen bonding interactions, and distances are indicated in angstroms. Color-coding for the atoms: carbon, green for receptor residues and gray for the ligand; nitrogen, blue; oxygen, red.

 

Three mutations (S151A, S152A, and K283A) were made in amino acids that were anticipated to establish favorable contacts with the {alpha} carboxyl group of the lysine ligand. Lysine 283 aligns with glutamate 292 in the binding pocket of mGluR1. In the crystal structure of mGluR1 it was suggested that this residue makes an indirect interaction via a water molecule with the {alpha} carboxyl group of the bound glutamate ligand (19). However, mutation of this residue caused a reduction in the binding of [3H]quisqualic acid, but little if any effects were seen in functional assays (33). Our results indicate that the equivalent amino acid, lysine 283, is an essential requirement for ligand binding and agonist activation of the 5.24 receptor and that it establishes a favorable contact with the {alpha} carboxyl group of the bound amino acid ligand (Fig. 7).

Serines 151 and 152 are conserved in the mGluR1 structure where they mediate hydrogen bonding with the {alpha} carboxyl group of the bound glutamate. However, mutation of either serine in the 5.24 receptor produced relatively little effect on [L-3H]lysine binding. The small effect on radioligand binding with the S152A mutant is particularly surprising, because this serine is highly conserved in most family 3 receptors, and in all previous instances where this serine was mutated in the mGluRs (23, 24, 33-35), or the GABAB-R1 subunit of the GABAB receptor (36), major losses or elimination of ligand binding and/or receptor function were observed. Surprisingly, in contrast to the small effects on binding, the S152A mutant displayed a 25-fold reduction in affinity for L-lysine in the functional assay, which measured receptor-induced release of intracellular calcium. The discrepancy between the effects on ligand binding and receptor activation suggest that, although this conserved serine plays a minor role in ligand recognition in the 5.24 receptor, it does appear to be critical for promoting the conformational change required for transmitting extracellular signals to the transmembrane domains and activation of the phosphoinositide/calcium signaling pathway.

Large reductions or elimination of binding and receptor activation were seen in mutations with all four amino acids projected to interact with the {alpha} amino group of the bound L-lysine (T175A, D195A, Y223A, and D309A). The molecular model indicated that the side chains of threonine 175, tyrosine 223, and aspartate 309 are positioned within close proximity (2.6-2.9 Å) to the {alpha} amino group of the docked amino acid ligand, whereas aspartate 195 is located further back (5.6 Å, Fig. 7). Thus rather than establishing a direct interaction with the ligand, the carboxyl side chain of Asp-195 may form a hydrogen bond with the hydroxyl group on the side chain of threonine 175, the orientation of which is critical for making a direct contact with the {alpha} amino group of the ligand.

These four residues are conserved in all eight mGluR receptor subtypes. Previous mutagenesis studies have demonstrated that the residues equivalent to Asp-195, Tyr-223, and Asp-309 are also obligatory for ligand binding to the mGluRs. The tyrosine analogous to Tyr-223 and the aspartate analogous to Asp-309 in the 5.24 receptor eliminated [3H]LY354740 binding to mGluR2 (34), and the aspartate equivalent to Asp-195 in the 5.24 receptor eliminated [3H]DCG-IV binding to mGluR3 (37). Moreover, it has also been shown that in mGluR8 the residues analogous to tyrosine 223 and aspartate 309 in the 5.24 receptor are critical for mediating the inhibitory properties of competitive antagonists (38). Intriguingly, five of the conserved amino acids that form bonds with the {alpha} amino and {alpha} carboxyl groups of L-lysine and L-glutamate in the 5.24 receptor and the mGluRs are also conserved in the CaR either as identical amino acids or as conservative substitutions (see Fig. 1 and Table II). Previous work has demonstrated that mutation of the rat CaR at amino acids corresponding to serine 152 and threonine 175 in the 5.24 receptor severely reduced the ability of calcium to activate the receptor (39). In addition, mutations at tyrosine 218 and glutamate 297 in the human CaR gene corresponding to tyrosine 223 and aspartate 309 in the 5.24 receptor cause the autosomal dominant genetic disease familial hypocalciuric hypercalcemia in humans in which the responsiveness of the CaR in the parathyroid gland and kidneys is impaired (40). Pertinent to this issue is the finding that activation of the CaR by calcium is enhanced in the presence of amino acids, particularly aromatic amino acids (31). Recently, it has been shown that serine 170 in the CaR (equivalent to threonine 175 in the 5.24 receptor) is required for amino acid modulation; it was suggested that the amino acid binding site in the CaR is close to or structurally dependent upon the calcium binding site of the receptor (41). These findings together with other experimental data indicating that calcium can activate some subtypes of mGluRs (42; also see Ref. 43) suggest a probable, but as yet incompletely understood relationship between the presence of cations and the binding of amino acids in family 3 receptors.

Several rodent and human taste receptors have been identified that respond to sweet and bitter substances and to amino acids. Heteromeric dimers of the T1R2 + T1R3 receptors are activated by sweet tasting compounds, whereas the T1R1 + T1R3 receptor combination responds to L amino acids (44, 45). A sequence alignment of the TR1 subclass of taste receptors and the mGluRs reveals an interesting pattern of sequence conservation. Curiously, of the four key residues that interact with the {alpha} amino groups of the glutamate and lysine ligands in the mGluRs and the 5.24 receptor, all four are conserved in the T1R1 and T1R3 amino acid-activated taste receptors, whereas only two of these four are conserved in the T1R2 receptor required for sensing sweet tasting substances. This pattern of receptor sensitivity is consistent with our results on the 5.24 receptor and further reinforces the postulate that the presence of the four highly conserved residues contacting the {alpha} amino group of the ligands is a characteristic signature of amino acid binding across different classes of receptors with widely varying physiological functions. From an evolutionary perspective, the residues that establish bonds with the {alpha} amino groups of L-lysine, L-glutamate, and aromatic amino acids in the 5.24 receptor, mGluRs, and CaR, respectively, are most likely an ancient structural feature that has been retained as an essential component of an amino acid recognition motif in family 3 G-protein-coupled receptors.


   FOOTNOTES
 
* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes for Health Research, and by the resources at the Molecular Design and Information Technology Centre, University of Toronto. 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: Dept. of Pharmaceutical Sciences, University of Toronto, 19 Russell St., Toronto, Ontario M5S 2S2, Canada. Tel.: 416-978-4494; Fax: 416-978-8511; E-mail: d.hampson{at}utoronto.ca.

1 The abbreviations used are: CaR, calcium-sensing receptor; CMV, cytomegalovirus; HEK, human embryonic kidney cells; mGluR, metabotropic glutamate receptor; PNGase F, peptide N-glycosidase F; MG-132, Z-Leu-Leu-Leu-aldehyde. Back


   ACKNOWLEDGMENTS
 
We thank Dr. J. Ngai for kindly providing the goldfish 5.24 receptor cDNA, E. Rosemond for assistance, and Drs. P. J. O'Brien and J. W. Wells for comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mombaerts, P. (1999) Science 286, 707-711[Abstract/Free Full Text]
  2. Takeda, S., Kadoaki, S., Haga, T., Takaesu, H., and Mitaku, S. (2002) FEBS Lett. 520, 97-101[CrossRef][Medline] [Order article via Infotrieve]
  3. Fredriksson, R., Lagerstrom, M. C., Lundin, L.-G., and Schioth, H. G. (2003) Mol. Pharmacol. 63, 1256-1272[Abstract/Free Full Text]
  4. Speca, D. J., Lin, D. M., Sorensen, P. W., Isacoff, E. Y., Ngai, J., and Dittman, A. H. (1999) Neuron 23, 487-498[CrossRef][Medline] [Order article via Infotrieve]
  5. Dulac, C., and Axel, R. (1995) Cell 83, 195-206[CrossRef][Medline] [Order article via Infotrieve]
  6. Matsunami, H., and Buck, L. B. (1997) Cell 90, 775-784[CrossRef][Medline] [Order article via Infotrieve]
  7. Ryba, N. J. P., and Tirindelli, R. (1997) Neuron 19, 371-379[CrossRef][Medline] [Order article via Infotrieve]
  8. Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M., Lytton, J., and Herbert, S. C. (1993) Nature 366, 575-580[CrossRef][Medline] [Order article via Infotrieve]
  9. Garrett, J. E., Capuano, I. V., Hammerland, L. G., Hung, B. C. P., Brown, E. M., Hebert, S. C., Nemeth, E. F., and Fuller, F. (1995) J. Biol. Chem. 270, 12919-12925[Abstract/Free Full Text]
  10. Ruat, M., Molliver, M. E., Snowman, A. M., and Snyder, S. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3161-3165[Abstract/Free Full Text]
  11. Hoon, M. A., Adler, E., Lindemeier, J., Battey, J. F., Ryba, N. J. P., and Zuker, C. S. (1999) Cell 96, 541-551[CrossRef][Medline] [Order article via Infotrieve]
  12. Chaudhari, N., Landin, A. M., and Roper, S. D. (2000) Nat. Neurosci. 3, 113-119[CrossRef][Medline] [Order article via Infotrieve]
  13. Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R., and Nakaniski, S. (1992) Neuron 8, 169-179[CrossRef][Medline] [Order article via Infotrieve]
  14. Kang, J., and Caprio, J. (1995) J. Neurophysiol. 73, 172-177[Abstract/Free Full Text]
  15. Nearing, J., Betka, M., Nentschel, H., Elger, M., Baum, M., Bai, M., Chattopadyhay, N., Brown, E. M., Herbert, S. C., and Harris, H. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9231-9236[Abstract/Free Full Text]
  16. Romano, C., Yang, W.-L., and O'Malley, K. L. (1996) J. Biol. Chem. 271, 28612-28616[Abstract/Free Full Text]
  17. Romano, C., Miller, J. K., Hyre, K., Dikranian, S., Mennerick, S., Takeuchi, Y., Goldberg, M. P., and O'Malley, K. L. (2001) Mol. Pharmacol. 59, 46-53[Abstract/Free Full Text]
  18. Pace, A. J., Gama, L., and Breitwieser, G. E. (1999) J. Biol. Chem. 274, 11629-11634[Abstract/Free Full Text]
  19. Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971-977[CrossRef][Medline] [Order article via Infotrieve]
  20. Okamoto, T., Sekiyama, N., Otsu, M., Shimada, Y., Sato, A., Nakanishi, S., and Jingami, H. (1998) J. Biol. Chem. 273, 13089-13096[Abstract/Free Full Text]
  21. Han, G., and Hampson, D. R. (1999) J. Biol. Chem. 274, 10008-10013[Abstract/Free Full Text]
  22. Peltekova, V., Han, G., Soleymanlou, N., and Hampson, D. R. (2000) Mol. Brain Res. 76, 180-190[Medline] [Order article via Infotrieve]
  23. O'Hara, P. J., Sheppard, P., Thogersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) Neuron 11, 41-52[CrossRef][Medline] [Order article via Infotrieve]
  24. Hampson, D. R., Huang, X.-P., Pekhletski, R., Peltekova, V., Hornby, G., Thomsen, C., and Thogersen, H. (1999) J. Biol. Chem. 274, 33488-33495[Abstract/Free Full Text]
  25. Costantino, G., Macchiarulo, A., and Pellicciari, R. (1999) J. Med. Chem. 42, 5390-5401[CrossRef][Medline] [Order article via Infotrieve]
  26. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779-815[CrossRef][Medline] [Order article via Infotrieve]
  27. Ramachandran, G. N., Ramakrishnan, C., and Sasisekharan, V. (1963) J. Mol. Biol. 7, 95-99[Medline] [Order article via Infotrieve]
  28. Case, D. A., Pearlman, D. A., Caldwell, J. W., Cheatham, T. E., III, Wang, J., Ross, W. S., Simmerling, C., Darden, T., Merz, K. M., Stanton, R. V., Cheng, A., Vincent, J. J., Crowley, M., Tsui, V., Gohlke, H., Radmer, R., Duan, Y., Pitera, J., Massova, I., Seibel, G. L., Singh, U. H., Weiner, P., and Kollman, P. A. (2001) AMBER, version 7.0, University of California, San Francisco, CA
  29. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Jr., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) J. Am. Chem. Soc. 117, 5179-5197[CrossRef]
  30. Kuang, D., and Hampson, D. R. (2002) Soc. Neurosci., Abstr. 247.5
  31. Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819[Abstract/Free Full Text]
  32. Bertrand, H. O., Bessis, A. S., Pin, J. P., and Acher, F. C. (2002) J. Med. Chem. 45, 3171-3183[CrossRef][Medline] [Order article via Infotrieve]
  33. Sato, T., Shimada, Y., Nagasawa, N., Nakanishi, S., and Jingami, H. (2003) J. Biol. Chem. 278, 4314-4321[Abstract/Free Full Text]
  34. Malherbe, P., Knoflach, F., Broger, C., Ohresser, S., Kratzeisen, C., Adam, G., Stadler, H., Kemp, J. A., and Mutel, V. (2001) Mol. Pharmacol. 60, 944-954[Abstract/Free Full Text]
  35. Rosemond, E., Peltekova, V., Naples, M., Thogersen, H., and Hampson, D. R. (2002) J. Biol. Chem. 277, 7333-7340[Abstract/Free Full Text]
  36. Galvez, T., Prezeau, L., Milioti, G., Franek, M., Joly, C., Froestl, W., Bettler, B., Bertrand, H. O., Blahos, J, and Pin, J. P. (2000) J. Biol. Chem. 275, 41166-41174[Abstract/Free Full Text]
  37. Yao, Y., Pattabiraman, N., Michne, W. F., Huang, X.-P., and Hampson, D. R. (2003) J. Neurochem. 86, 947-957[CrossRef][Medline] [Order article via Infotrieve]
  38. Bessis, A. S., Rondard, P., Gaven, F., Brabet, I., Triballeau, N., Prezeau, L., Acher, F., and Pin, J. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11097-11102[Abstract/Free Full Text]
  39. Brauner-Osborne, H., Jensen, A. A., Sheppard, P. O., O'Hara, P., and Krogsgaard-Larsen, P. (1999) J. Biol. Chem. 274, 18382-18386[Abstract/Free Full Text]
  40. Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, S. H. S., Pollak, M. R., Krapcho, K., Herbert, S. C., and Brown, E. M. (1996) J. Biol. Chem. 271, 19537-19545[Abstract/Free Full Text]
  41. Zhang, Z., Qiu, W., Quinn, S. J., Conigrave, A. D., Brown, E. M., and Bai, M. (2002) J. Biol. Chem. 277, 33727-33735[Abstract/Free Full Text]
  42. Kubo, Y., Miyashita, T., and Murata, Y. (1998) Science 279, 1722-1725[Abstract/Free Full Text]
  43. Nash, M. S., Saunders, R., Young, K. W., Challiss, R. A. J., and Nahorski, S. R. (2001) J. Biol. Chem. 276, 19286-19293[Abstract/Free Full Text]
  44. Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M., and Adler, E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4692-4696[Abstract/Free Full Text]
  45. Nelson, G., Chandrashekar, J., Hoon, M. A., Feng, L., Zhao, G., Ryba, N. J. P., and Zuker, C. S. (2002) Nature 416, 199-202[CrossRef][Medline] [Order article via Infotrieve]

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