Construction of a High Affinity Zinc Binding Site in the Metabotropic Glutamate Receptor mGluR1

NONCOMPETITIVE ANTAGONISM ORIGINATING FROM THE AMINO-TERMINAL DOMAIN OF A FAMILY C G-PROTEIN-COUPLED RECEPTOR*

Anders A. JensenDagger , Paul O. Sheppard§, Liselotte B. Jensen, Patrick J. O'Hara§, and Hans Bräuner-OsborneDagger ||

From the NeuroScience PharmaBiotec Research Centre, Departments of Dagger  Medicinal Chemistry and  Pharmacology, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark, and § ZymoGenetics Inc., Seattle, Washington 98102

Received for publication, August 9, 2000, and in revised form, December 21, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn2+ binding site in mGluR1b by mutating the residue Lys260 to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC50 value of 2 µM. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn2+ to antagonize the agonist-induced response. Zn2+ binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

(S)-Glutamic acid (Glu)1 is the major excitatory neurotransmitter in the central nervous system and plays an essential role in a wide range of functions, such as brain development, memory formation, and neurotoxicity (1). Excessive glutamatergic signaling has been linked to the pathogenesis of certain psychiatric and neurological disorders, including schizophrenia, epilepsy, and ischemia (1). Glu mediates its effects through two distinct types of receptors: the ionotropic glutamate receptors and the metabotropic glutamate receptors (mGluRs). Whereas the ionotropic glutamate receptors form ligand-gated ion channels, which directly mediate electrical signaling of nerve cells, the G-protein-coupled mGluRs regulate signaling through stimulation or inhibition of intracellular metabolic cascades (1).

The mGluRs are members of family C of the G-protein-coupled receptor (GPCR) superfamily, which also includes two gamma -aminobutyric acid receptors type B (GABABR1-2) (2), a calcium-sensing receptor (CaR) (3), a family of putative pheromone receptors (4), and three recently cloned orphan receptors (5-7). Molecular cloning has revealed the existence of eight different mGluR subtypes, subdivided into three subgroups based on their amino acid sequence similarities, agonist pharmacology, and signal transduction pathways (1, 8-10). Group I is constituted by mGluR1 and mGluR5, group II by mGluR2 and mGluR3, and group III by the remaining four subtypes. Some of the subtypes exist as multiple splice variants, which only differ in their carboxyl termini (10). In the case of mGluR1, the splice variant mGluR1a possesses a C-terminal of 359 amino acid residues, whereas the termini of the other splice variants cloned so far only consist of 50-60 residues.

With the exception of the orphan receptors in family C (5-7), all of its members contain remarkably large extracellular amino-terminal domains (ATDs). The ATDs can be up to 600 amino acids long, and agonist binding to the family C receptor has been demonstrated to take place to this region (11-17). Weak amino acid sequence similarities exist between these ATDs and a family of periplasmic binding proteins (PBPs) involved in the transfer of nutrients into the cytoplasm in bacteria (11). We have previously applied the crystal structure of one of these PBPs, the leucine/isoleucine/valine-binding protein, as a template for the construction of a tertiary molecular model of the ATD of mGluR1, and similar models have been published for mGluR4, GABABR1, and CaR (11, 18-20). According to these models, the ATD of the family C receptor consists of two globular domains ("lobes") connected by a hinge region. Based on the information obtained from crystal structures of the PBPs, the ATD of the family C receptor is speculated to exist in two conformations: an "open" inactivated and a "closed" activated form (21). Agonist binding to the ATD is believed to take place to a closed form of the region. How the closure of ATD subsequently is translated into G-protein coupling and effector enzyme activation or inhibition is at present poorly understood.

Two conserved serine/threonine residues throughout the PBPs and the family C receptors have been shown to be important for agonist binding to and/or activation of mGluR1, mGluR4, GABABR1, and CaR (11, 16, 18, 19). Furthermore, an arginine residue conserved within the mGluRs has been shown to be crucial for agonist binding to and/or activation of mGluR1 and mGluR4, most likely through an ionic interaction with the distal negatively charged group of the agonists (18, 22). According to our model of the "open" ATD of mGluR1, the three residues identified as key participants in agonist binding, Arg78, Ser165, and Thr188, are located in the same lobe of the ATD.

As an attempt to shed light on the contraction process of the ATD following agonist binding, we have created a high affinity zinc binding site in mGluR1 by mutating Lys260, located in the top of the lobe opposite to the lobe containing the agonist binding residues, to a histidine. The results of the study provide interesting information regarding the ATD closure mechanism and the structural flexibility within each ATD lobe.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Culture media, serum, antibiotics, and buffers for cell culture were obtained from Life Technologies (Paisley, UK). [3H]Quisqualic acid ([3H]Quis) and myo-[2-3H]inositol were purchased from Amersham Pharmacia Biotech. (S)-4-Carboxyphenylglycine ((S)-4CPG) and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) were purchased from Tocris (Bristol, UK). Rabbit anti-mGluR1a polyclonal antibody and horseradish peroxidase-goat anti-rabbit IgG were purchased from Chemicon (Temecula, CA) and Zymed Laboratories Inc. (San Francisco, CA), respectively. All other chemicals were obtained from Sigma. The pSI vector was obtained from Promega (Madison, WI). The plasmids pmGluR1a, rCaR-pRK5, and m5-pCD were generous gifts from Professor Shigetada Nakanishi (Kyoto University, Japan), Professor Solomon H. Snyder (The John Hopkins University School of Medicine, Baltimore, MD), and Dr. Mark R. Brann (ACADIA Pharmaceuticals, San Diego, CA), respectively. The tsA cells and the mGluR1a-CHO cell line were generous gifts from Dr. Penelope S. V. Jones (University of California, San Diego, CA) and professor Shigetada Nakanishi, respectively.

Subcloning of Receptors and Construction of Mutant Receptors-- The subcloning of mGluR1b, mGluR1a, and CaR from their respective vectors into the pSI vector have all been described previously (16, 23). The point-mutated mGlu1b receptors were constructed using the QuickChange mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The construction of mutant K260H mGluR1a-pSI for the Western blot experiments were done by subcloning a region containing the Lys260 right-arrow His mutation from mutant K260H mGluR1b-pSI into mGluR1a-pSI using the unique restriction enzymes XhoI and ApaI. All amplified receptor DNAs were sequenced on an ABI 310 sequencer using the Big Dye Terminator Cycle Sequencing kit (PerkinElmer Life Sciences).

Cell Culture-- tsA cells (a transformed HEK 293 cell line (24)) were maintained at 37 °C in a humidified 5% CO2 incubator in Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% calf serum.

Inositol Phosphate (IP) Assay-- 1 × 106 tsA cells were split into a 10-cm tissue culture plate and transfected with 5 µg of plasmid the following day using Superfect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany). The day after transfection, the cells were split into a poly-D-lysine-coated 24-well tissue culture plate in inositol-free Dulbecco's modified Eagle's medium, supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), 10% dialyzed fetal calf serum, and 1 µCi/ml myo-[2-3H]inositol. 16-24 h later, the cells were washed with Hanks' balanced saline solution (HBSS) and incubated at 37 °C for 10 min in HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, and 10 mM LiCl (and in the antagonism experiments, various concentrations of ZnCl2 or 100 µM CPCCOEt). The buffer was removed, and the cells were incubated for 40 min in the same buffer containing various concentrations of Glu, a fixed Glu concentration and various concentrations of ZnCl2 or 100 µM CPCCOEt, or containing a fixed ZnCl2 concentration and various concentrations of Glu. The reactions were stopped by exchanging the buffer with 500 µl of ice-cold 20 mM formic acid, and separation of total [3H]inositol phosphates was carried out by ion exchange chromatography as described previously (23, 25, 26). The effect of ZnCl2 on tsA cells transfected with CaR-pSI or m5-pCD was studied in the same manner, except that CaCl2 and acetylcholine were used as agonists, respectively. The effects of CuCl2 and CoCl2 on cells transfected with wild type (WT) or K260H mGluR1b were studied using the same protocol as in the Zn2+ antagonism experiments. Almost all IP experiments were performed in triplicate (a few were performed in duplicate), and the results are given as mean ± S.E. of at least three independent experiments.

[3H]Quis Binding Assays-- 2 × 106 tsA cells were split into a 15-cm tissue culture plate and transfected with 10 µg of plasmid the following day using Superfect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany). The day after transfection, the medium was changed to Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% dialyzed fetal calf serum. The following day, the cells were suspended in ice-cold 20 mM HEPES-NaOH, 10 mM EDTA (pH 7.4) and centrifuged at 50,000 × g at 4 °C for 20 min. The pellet was homogenized (using a Polytron homogenizer for 10 s) in ice-cold 20 mM HEPES-NaOH, 0.1 mM EDTA (pH 7.4) and centrifuged at 50,000 × g at 4 °C for 20 min. This step was performed twice. The membranes were homogenized in assay buffer, 20 mM HEPES-NaOH, 2 mM MgCl2, 2 mM CaCl2 (pH 7.4), and protein concentrations were measured using Bradford Protein Assay with bovine serum albumin as standard (Bio-Rad). In the saturation binding experiments with WT, K260H, and K260H/H231A/E233A/E238A mGluR1b, 5-15 µg of membrane fractions were incubated for 1 h at room temperature with various concentrations of [3H]Quis in the absence or presence of 1 mM Glu (representing total and nonspecific binding, respectively) in a total assay volume of 100 µl. [3H]Quis was diluted with unlabeled (S)-quisqualic acid at concentrations above 250 nM. Expression levels of other mutant receptors were estimated by measuring binding at a concentration of 250 nM [3H]Quis in the absence and presence of 1 mM Glu. To investigate whether Zn2+ was able to displace [3H]Quis binding to WT or K260H mGluR1b, 50-100-µg membrane fractions of cells transfected with the two receptors were incubated for 1 h at room temperature with 30 nM [3H]Quis in the absence or presence of 1 mM Glu, 100 µM (S)-4CPG, 100 µM CPCCOEt, or 100 µM ZnCl2. The assay mixture was aspirated onto a GF/C filter (Whatman) and washed with 3 × 5 ml of ice-cold assay buffer, after which the filters were dried and counted in a scintillation counter. The binding experiments were performed in duplicate or triplicate at least three times.

Western Blots-- The rabbit anti-mGluR1a polyclonal antibody used is directed toward the Glu1116-Leu1130 peptide in the C terminus of mGluR1a. This region does not exist in the "short tailed" splice variant mGluR1b. Hence, we used WT mGluR1a and the mutant K260H mGluR1a in the experiments. 1 × 106 tsA cells were split into a 10-cm tissue culture plate and transfected with 5 µg of plasmid (WT mGluR1a-pSI, K260H mGluR1a-pSI, or pSI) the following day using Superfect as a DNA carrier according to the protocol by the manufacturer (Qiagen, Hilden, Germany). The day after transfection, the medium was changed to Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% dialyzed fetal calf serum. The following day, the cells were washed twice in HBSS and scraped into lysis buffer (2 mM HEPES, 2 mM EDTA, containing protease inhibitors (CompleteTM, Roche, Switzerland) and 100 mM iodoacetamide) and homogenized. After centrifugation at 1000 × g for 5 min, the nuclear pellet was discarded, and membranes were harvested by centrifugation at 35,000 × g for 30 min. The membranes were homogenized in HBSS containing 1% SDS and 100 mM iodoacetamide and incubated for 30 min in the presence or absence of 300 mM beta -mercaptoethanol, 100 mM dithiothreitol, or 10 mM ZnCl2. The samples were heated for 5 min at 65 °C before electrophoresis, where the samples (5-15 µg of membrane protein/well) were separated on 7.5% polyacrylamide gels. The proteins were transferred to nitrocellulose membranes by transblotting at 200 mA for 1 h. The blots were incubated in phosphate-buffered saline solution (PBS) containing 0.05% Tween 20 and 2% powdered skim milk overnight. The blots were washed three times for 30 min with PBS containing 0.05% Tween 20 and incubated with rabbit anti-mGluR1a polyclonal antibody (diluted 1:2000 in PBS containing 0.05% Tween 20 and 2% powdered skim milk) for 1 h. This was followed by three 30-min washes in PBS containing 0.05% Tween 20 and incubation in anti-Rabbit IgG conjugated to horseradish peroxidase (diluted 1:1500 in PBS containing 0.05% Tween 20 and 2% powdered skim milk) for 1 h, after which the membranes were developed in a solution of 3,3-diaminobenzidine (Kem-En-Tec A/S, Copenhagen, Denmark). Three independent Western blot experiments were performed, and similar results were obtained in all of them.

Data Analysis-- Data from Glu concentration-response experiments were fitted to the following simple mass equation: R = Rbasal + (Rmax/(1 + (EC50/[A]n))), where [A] is the concentration of agonist, n is the Hill coefficient, and R is the response. Data from the Zn2+ antagonism experiments were fitted to the following: R = Rmax - (Rmax/(1 + (IC50/[A]n))), where [A] is the concentration of antagonist, n is the Hill coefficient, and R is the response. Curves were generated by nonweighted least-squares fits using the program KaleidaGraph 3.08 (Synergy Software, Reading, PA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Zn2+ on WT mGluR1b-- Exposure of tsA cells transiently transfected with WT mGluR1b to Zn2+ in concentrations of 300 µM and above led to an inhibition of agonist-induced IP accumulation with an IC50 value of the metal ion of ~1 mM (Fig. 1). However, similar pharmacological profiles were observed when two other phosholipase C-coupled receptors, CaR and the muscarinic acetylcholine receptor m5, were exposed to Zn2+ (Fig. 1). The observation that zinc displays the same apparent antagonistic effect at three different receptors strongly suggests that the observed inhibition is of a nonspecific nature. Zn2+ appears to have an inhibitory or a toxic effect on the tsA cells. Alternatively, this ion may inhibit events downstream in the signal transduction pathway of the receptors. In agreement with the findings in this study, the group of Pin have also only observed a minute decrease in the maximal response (induced by 1 mM Glu) exerted by 100 µM ZnCl2 on mGluR1.2 Since the nonspecific effect of Zn2+ on receptor functionality in our assay is negligible at concentrations below 300 µM, 100 µM was used as the maximal concentration of Zn2+, when testing the WT and mutant mGlu1b receptors.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   The effects of Zn2+ on agonist-induced IP accumulation in tsA cells transiently transfected with mGluR1b-pSI, CaR-pSI, or m5-pCD. The IP assays were performed as described under "Experimental Procedures." The cells were prelabeled overnight with 1 µCi/ml myo-[2-3H]inositol, washed with HBSS, and incubated for 10 min with HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2. The buffer was removed, and the cells were incubated for 40 min in the same buffer containing agonist (1 mM Glu, 3 mM CaCl2, or 50 nM acetylcholine, respectively) and various concentrations of ZnCl2. The buffer was aspirated, and the reactions were stopped by the addition of ice-cold 20 mM formic acid. Total IP formation was determined by an ion exchange assay. Data are given as percentage of the IP accumulation in the absence of ZnCl2.

Effects of Zn2+ on mutant mGluR1bs-- In the experiments of Zn2+ antagonism of Glu-induced receptor activation, an agonist concentration of 1 mM Glu was used. At this concentration, the WT and all mutant receptors were fully activated. In cells transfected with mutant K260H mGluR1b, where Lys260 had been substituted with a histidine, the IC50 of Zn2+ was decreased 500-fold compared with the "apparent" IC50 of this ion on the WT receptor (Fig. 2 and Table I). Zn2+ showed an affinity on the mutant receptor independent of the agonist concentration used, indicating a noncompetitive antagonist effect (Fig. 3A). This proposal was supported by a Schild analysis, where Glu concentration-response experiments were performed in the presence of increasing concentrations of Zn2+. Here, the maximal response of K260H was markedly reduced with increasing concentrations of Zn2+ present, whereas the EC50 values of Glu were relatively unaltered (Fig. 3B). Furthermore, much like the noncompetitive mGluR1 antagonist CPCCOEt and in contrast to the competitive antagonist (S)-4CPG, Zn2+ did not influence [3H]Quis binding to either WT or K260H mGluR1b (Fig. 4A). In conclusion, Zn2+ was clearly a noncompetitive antagonist at mutant K260H. The metal ions Cu2+ and Co2+ were tested on WT and K260H mGluR1b. However, CuCl2 and CoCl2 were unable to antagonize the agonist-induced response in both the WT and the mutant receptor in concentrations up to 100 µM (data not shown). In concentrations above 100 µM, CuCl2 had a marked toxic effect on the tsA cells.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition by ZnCl2 of agonist-induced IP accumulation in tSA cells transiently transfected with WT and mutant mGluR1b receptors. The IP assays were performed as described under "Experimental Procedures." The cells were prelabeled overnight with 1 µCi/ml myo-[2-3H]inositol, washed with HBSS, and incubated for 10 min with HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2 or 100 µM CPCCOEt. The buffer was removed, and the cells were incubated for 40 min in the same buffer containing 1 mM Glu and various concentrations of ZnCl2 or 100 µM CPCCOEt. The buffer was aspirated, and the reactions were stopped by the addition of ice-cold 20 mM formic acid. Total IP formation was determined by an ion exchange assay. Data (mean ± S.D.) are given as percentage of the difference of the response in absence of antagonist and the response in the presence of 100 µM CPCCOEt.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Pharmacological characterization of WT and mutant mGlu1b receptors
Shown are the pharmacological parameters of WT and selected mutant mGlu1b receptors from Glu concentration-response experiments and Zn2+ inhibition of Glu-induced IP accumulation. The IP assays were performed as described under "Experimental Procedures" and in the legends to Figs. 2 and 6, and the data are given as mean ± S.E. of at least three independent experiments.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Zn2+ is a noncompetitive antagonist on K260H mGluR1b. The IP assays were performed as described under "Experimental Procedures." A, inhibition by ZnCl2 of IP accumulation induced by 3 µM, 50 µM, and 1000 µM Glu in tsA cells transiently transfected with K260H mGluR1b-pSI. The cells were prelabeled overnight with 1 µCi/ml myo-[2-3H]inositol, washed with HBSS, and incubated for 10 min with HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and various concentrations of ZnCl2. The buffer was removed, and the cells were incubated for 40 min in the same buffer containing three different concentrations of Glu (3, 50, or 1000 µM) and various concentrations of ZnCl2. The buffer was aspirated, and the reactions were stopped by the addition of ice-cold 20 mM formic acid. Total IP formation was determined by an ion-exchange assay. Data (mean ± S.D.) are given as percentage of the difference of the response in the absence and presence of 10 mM ZnCl2. B, Schild analysis of the inhibition by ZnCl2 of IP accumulation induced by Glu in tsA cells transiently transfected with K260H mGluR1b-pSI. The cells were prelabeled overnight with 1 µCi/ml myo-[2-3H]inositol, washed with HBSS, and incubated for 10 min with HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, 10 mM LiCl, and four different concentrations of ZnCl2 (0, 1, 4, or 16 µM). The buffer was removed, and the cells were incubated for 40 min in the same buffer containing the four different concentrations of ZnCl2 and various concentrations of Glu. The buffer was aspirated, and the reactions were stopped by the addition of ice-cold 20 mM formic acid. Total IP formation was determined by an ion exchange assay. Data are given as disintegrations/min (DPM) per well (mean ± S.D).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   [3H]Quis binding to WT and mutant mGlu1b receptors. Displacement of [3H]Quis binding to WT and K260H mGluR1b (A) and saturation binding to WT, K260H, and K260H/H231A/E233A/E238A mGluR1b are shown. A, 50 µg of membrane fractions of tsA cells transfected with WT (black bars) or K260H (gray bars) mGluR1b were prepared as described under "Experimental Procedures" and incubated at room temperature for 1 h with 30 nM [3H]Quis alone (Total) or in the presence of 1 mM Glu, 100 µM (S)-4CPG, 100 µM CPCCOEt, or 100 µM ZnCl2. The assay mixture was aspirated onto a GF/C filter, washed with 3 × 5 ml of ice-cold assay buffer, after which the filters were dried and counted in a scintillation counter. Data are given as disintegrations/min (DPM) per well (mean ± S.D.). B, 5-15 µg of membrane fractions of WT, K260H, or K260H/H231A/E233A/E238A mGluR1b transfected cells prepared as described under "Experimental Procedures" were incubated at room temperature for 1 h with various concentrations of [3H]Quis alone or in the presence of 1 mM Glu. [3H]Quis was diluted with unlabeled (S)-quisqualic acid at concentrations above 300 nM. The assay mixture was aspirated onto a GF/C filter, washed with 3 × 5 ml of ice-cold assay buffer, after which the filters were dried and counted in a scintillation counter. C, binding of 250 nM [3H]Quis to 5-7 µg of membrane fractions of WT and mutant mGluR1b-transfected cells were performed as described for B and under "Experimental Procedures" in the absence or presence of 1 mM Glu.

We wished to examine whether we had created a Zn2+ site in K260H that actually involved the introduced histidine or whether it was the removal of the positively charged lysine residue that had eliminated a repulsive obstruction, making Zn2+ binding to other amino acids in the region possible. Thus, we mutated Lys260 to an alanine, and the K260A mutant was shown to display a pharmacological profile similar to that of the WT receptor upon exposure to Zn2+ (Table I). Hence, the histidine introduced in K260H is in fact involved in and is essential for the Zn2+ binding to and antagonism of the K260H mutant.

To identify the amino acid(s) forming the Zn2+ binding site together with the introduced histidine in K260H, we mutated five amino acids, which, according to our model of the open ATD of mGluR1, are located in proximity to Lys260 (Fig. 5) (10). The five residues, His231, Glu233, Glu238, His257, and Asp259, are all potential "Zn2+ ligands" (27-29). As can be seen from Table I, single alanine mutations of these five residues in K260H did not reduce the antagonistic potency of Zn2+. Mutants with the Lys260 right-arrow His mutation and different combinations of two of these mutations were constructed as well, some of which are shown in Table I. However, none of these combinations of mutations impaired the ability of Zn2+ to antagonize the receptor significantly. In contrast, simultaneous mutation of the three residues His231, Glu233 and Glu238 (mutant K260H/H231A/E233A/E238A) resulted in a receptor displaying a pharmacological profile of Zn2+ not significantly different from that on the WT receptor, indicating that the binding of this ion had been eliminated (Fig. 2).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 5.   Detail from the molecular model of the open form of the ATD of mGluR1. Shown is a depiction of the upper part of the mGluR1 ATD lobe not involved in agonist binding. Lys260 and the regions potential Zn2+ coligands are shown. The construction of the model of the open form of the mGluR1 ATD has been published previously (11).

Substitution of the Participants in the Zinc Binding Site-- The possibility cannot be excluded that the loss of Zn2+ antagonism of mutant K260H/H231A/E233A/E238A as compared with mutant K260H is the result of indirect structural changes within the region, caused by the mutations. To study this, we substituted the three "coligands" (His231, Glu233, and Glu238) with other residues capable of coordinating the metal ion, a principle previously used by Gether and co-workers (30) in a study of an endogenous zinc site in the dopamine transporter. Interestingly, mutant K260H/H231E/E233H/E238H was not only still antagonized by zinc, the potency of the metal ion was in fact significantly increased as compared with K260H (Table I). The substitution of the three coligands appeared to have improved the binding of Zn2+ to the receptor.

Agonist Pharmacology of WT and Mutant mGluR1bs-- In agreement with a previous study of mGluR1b transiently expressed in tsA cells, the WT receptor displayed a concentration-dependent 3-4-fold increase in IP accumulation when exposed to Glu (Fig. 6 and Table I) (22). The pharmacological profiles of Glu on the majority of mutants tested were not significantly different from that on the WT receptor (Table I). However, mutants containing the Glu238 right-arrow Ala and, to a smaller extent, the Glu233 right-arrow Ala mutation displayed slightly elevated basal responses as compared with WT. Furthermore, mutation of Glu238 to histidine resulted in a further increased basal response (Fig. 6). The elevated basal activity could be reduced with both the competitive mGluR antagonist, (S)-4CPG, and the noncompetitive mGluR1 antagonist, CPCCOEt (data not shown). This is quite interesting considering the reported increased agonist affinities caused by an alanine mutation of Gln312 in the corresponding region of GABABR1a (19). The functional importance of Glu238 in mGluR1 is currently under investigation and will not be discussed further in this report.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Glu-induced IP accumulation in tsA cells transiently transfected with WT, K260H, or K260H/E238H mGluR1b. The IP assays were performed as described under "Experimental Procedures." The cells were prelabeled overnight with 1 µCi/ml myo-[2-3H]inositol, washed with HBSS, and incubated for 10 min with HBSS supplemented with 0.9 mM CaCl2, 1.05 mM MgCl2, and 10 mM LiCl. The buffer was removed, and the cells were incubated for 40 min in the same buffer containing various concentrations of Glu. The buffer was aspirated, and the reactions were stopped by the addition of ice-cold 20 mM formic acid. Total IP formation was determined by an ion exchange assay. Data are given as disintegrations/min (DPM) per well (mean ± S.D.).

To investigate whether the functional profiles of the mutant and WT receptors were mimicked in an agonist binding assay, we characterized [3H]Quis binding to WT, K260H, and K260H/H231A/E233A/E238A mGluR1b and estimated the expression levels of all mutants containing alanine substitutions of His231, Glu233, or Glu238 (Table II and Fig. 4). The Kd values of the radioligand at mutants K260H and K260H/H231A/E233A/E238A were not significantly different from that of the WT receptor (Fig. 4B and Table II). The expression levels of selected mutants were estimated by measuring the specific binding at 250 nM [3H]Quis. At this concentration, equilibrium is reached for the binding to WT, K260H, and K260H/H231A/E233A/E238A mGluR1b. The expression levels of all of the mutants tested were comparable with that of the WT receptor (Fig. 4C). Hence, the mutations in the ATD lobe did not appear to have affected agonist affinities or expression densities of the receptor.

                              
View this table:
[in this window]
[in a new window]
 
Table II
[3H]-Quis binding to WT and mutant mGluRlb receptors
The saturation binding assays were performed as described under "Experimental Procedures" and in the legends to Fig. 4, and the data are given as mean ± S.E. of at least three independent experiments.

Dimerization of WT and K260H mGluR1a-- The mGluRs have been demonstrated to exist as disulfide-linked homodimers (14, 15, 31). In a recent preliminary study of mGluR5 by Romano and co-workers (32), the cysteine residues Cys129 and Cys240 have been reported to be crucial for the covalent and noncovalent dimerization of the receptor, respectively. In agreement with studies of the functional importance of Cys140 in mGluR1 and of Cys129 and Cys131 in CaR, mutation of Cys129 in mGluR5 did not impair the pharmacology of the receptor significantly (20, 32, 33). In contrast, mutation of Cys240 in mGluR5 resulted in a nonfunctional receptor (32). Cys240 in mGluR5 is conserved in all of the mGluRs, and since the corresponding cysteine residue in mGluR1, Cys254, is located close to Lys260, the effects of Zn2+ on the dimerization process of WT and K260H mGluR1a were investigated by Western blotting, using an antibody directed toward the Glu1116-Leu1130 region in the C-terminal of rat mGluR1a (34). As can be seen from Fig. 7, the expression level of K260H appeared to be somewhat reduced compared with that of the WT receptor. This contrasts with the findings from [3H]Quis binding experiments, where the Bmax values obtained for WT and K260H mGluR1b were comparable in size (Fig. 4C and Table II). Under nonreducing conditions, both WT and K260H mGluR1a displayed two close bands at ~300 kDa. The smallest of the two bands disappeared in the presence of 300 mM beta -mercaptoethanol or 100 mM dithiothreitol, whereas the intensity of the larger band was not altered significantly. Under these reducing conditions, a band at 150 kDa corresponding to the monomeric receptor appeared. The size of the monomeric receptor is in excellent agreement with the predicted size of the recombinant receptor and with the findings of recent studies (35, 36). None of the oligomeric bands or the monomeric band were observed in membranes prepared from tsA cells transfected with the pSI vector (data not shown). An identical oligomeric/monomeric pattern and similar sizes of the oligomeric and monomeric bands were observed, when membranes prepared from the mGluR1a-CHO cell line were used (data not shown). To investigate whether the upper band could be ascribed to nonspecific receptor aggregation due to overexpression of the receptors on the tsA cell surface, the tsA cells were transfected with 1.5 µg of WT or K260H mGluR1a-pSI per 10-cm tissue culture plate (~30% of the cDNA used in Fig. 7). However, a Western blot of membranes prepared from these cells displayed the same two oligomeric bands under nonreducing conditions, and only the lower band was converted into a monomer band at 150 kDa in the presence of 300 mM beta -mercaptoethanol (data not shown). The only difference between this experiment and the experiment depicted in Fig. 7 was that the expression levels of both WT and K260H mGluR1a were markedly reduced. At present, a fully satisfactory explanation of the presence of two dimer/oligomer bands is not obvious. However, the oligomeric/monomeric pattern, where only one of two oligomeric bands was converted to a monomeric band upon exposure to a reducing agent, closely resembles that observed by two other groups in studies of the dimerization of mGluR1 and another family C receptor, CaR (36, 37).


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 7.   Imunoblotting analyses of WT and K260H mGluR1a-transfected tsA cells. 7 µg of membrane protein prepared from WT and K260H mGluR1a-pSI-transfected tsA cells were preincubated in the absence (lane A) or in the presence of 100 mM dithiothreitol (lane B), 300 mM beta -mercaptoethanol (lane C), or 10 mM ZnCl2 (lane D). The samples were separated on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and detected with a rabbit anti-mGluR1a polyclonal antibody as described under "Experimental Procedures." The left lane is a ladder, and the 150- and 250-kDa bands are given.

In any case, it is quite clear that Zn2+ has no effect on the oligomer/monomer ratio for either WT or K260H mGluR1a. In both cases, the oligomer bands were unaltered in their intensities, and no band was observed at 150 kDa in the presence of the metal ion in concentrations up to 10 mM (Fig. 7). In conclusion, zinc binding to the K260H mGluR1a mutant does not appear to interfere with the receptor-receptor interactions necessary for dimerization.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although key determinants for agonist binding to mGluR1 and the other mGluRs have been proposed (11, 18, 22), very little is known about the ATD contraction process following agonist binding to the receptor or the transference of the activation signal from the ATD to the G-protein coupling region. The agonist binding residues Arg78 and Ser165 as well as Thr188 are located in one of the two ATD lobes in mGluR1 (11, 22). Hence, the "closure" of the ATD may be a basic conformational change within this lobe having no consequences or minute consequences for the proximity of the two lobes. However, Galvez et al. (19) have recently demonstrated that alanine mutations of the residues Ser247 and Gln312 located on the "lip" of each ATD lobe in GABABR1a have a potentiating effect on agonist affinities. Based on their model of the ATD of GABABR1a, the authors suggested that these residues would be located close to each other in the closed ATD conformation of the receptor and that the mutations simply had improved the interaction between the lips of the two lobes (19). Furthermore, in a recent follow up study, it was shown that the residues in both lobes of the ATD of GABABR1a participate in the binding of the GABAB agonist baclofen (38). Clearly, these observations suggest a much more drastic structural change throughout the entire ATD region of the family C receptor bringing the lips as well as other parts of the two lobes in close proximity.

Creation of metal ion binding sites in GPCRs was introduced by Schwartz and co-workers, and the technique has been used to gain structural information about the spatial orientation of transmembrane regions (TMs) in family A and B GPCRs and well as other transmembrane proteins (30, 39-46). In the present study, we decided to explore the structural flexibility within the ATD lobe opposite to the lobe involved in agonist binding to mGluR1 and the implications of an introduced structural constraint in this lobe for receptor functionality. Hence, we constructed a zinc site in the upper part of this lobe by mutating Lys260 to a histidine. According to our previously published model of the open form of the ATD of mGluR1 (11), the lysine residue is located in close proximity to several histidine, glutamate, and aspartate residues (Fig. 5). Since it is well established from protein crystal structures that histidines, cysteines, glutamates, and aspartates are the prevailing ligands for Zn2+ in proteins (27-29), some of these residues were supposed to function as coligands for Zn2+ in the constructed mutant. Zn2+ actually turned out to be a quite potent noncompetitive antagonist of agonist-induced IP accumulation on mutant K260H, whereas it had no measurable effects on the response of the WT receptor or the mutant K260A at the Zn2+ concentrations used. The inability of Zn2+ to antagonize the agonist response of K260A clearly demonstrates that the histidine introduced in mutant K260H is an essential participant in the binding of Zn2+. His231, Glu233, and Glu238 were identified as other residues involved in the binding of this ion. This is in excellent agreement with their location and spatial orientation in our model of the open ATD of K260H mGluR1 (Fig. 5). His231, Glu233, and Glu238 are located on the same strand in the ATD lobe, whereas the histidine introduced instead of Lys260 is located on another. Each of the three "coligands" are located ~6 Å from the introduced histidine. Considering that the distances in metal ion-histidine and bidentate metal ion-carboxylate interactions in proteins typically are 2-2.5 Å (27, 29, 47), Zn2+ binding between the introduced histidine and these three residues seems feasible.

Removal of ligands from the tridentate zinc binding sites in enzymes and transmembrane proteins and mutants of these have been demonstrated to lead to decreased zinc affinities of the metal ion (30, 39, 48, 49). Thus, it is surprising that all three endogenous Zn2+ ligands in K260H mGluR1b (His231, Glu233, and Glu238) had to be mutated to alanines to eliminate Zn2+ binding to the receptor. The typical coordination arrangement of Zn2+ binding to proteins is tetrahedral or distorted tetrahedral, where the protein most often contributes with three or four ligands to the binding (27-29). However, zinc and other metal ions are able to coordinate to proteins in a bidentate manner, with solvent molecules acting as the remaining ligands (29, 50-52), and several bidentate zinc sites have been introduced in the TMs of GPCRs (39, 40, 42-46). Considering that the residues involved in zinc binding to K260H mGluR1 are located in the top of the ATD lobe, the region would be expected to be solvent-accessible. The idea that Zn2+ is capable of binding to the introduced histidine and three different coligands in K260H mGluR1 with roughly the same affinity is still quite remarkable. However, a histidine introduced instead of Glu193 in TM5 of the tachykinin NK-1 receptor has been shown to be able to participate in two different bis-histidine sites, which display comparable affinities of Zn2+, by coordinating to introduced histidines in TM3 (Asn109 right-arrow His) or TM6 (Tyr272 right-arrow His) as coligands (39, 40). Clearly, some degree of flexibility in the orientation of the introduced histidine in TM5 has to exist to achieve binding to both TMs. Since the strands in the ATD lip of mGluR1 in contrast to the transmembrane alpha -helices are not constricted by the surrounding plasma membrane, they probably are more flexible in their intramolecular orientation. Hence, the idea of bidentate Zn2+ sites between the introduced histidine in K260H and each of the three coligands must be considered a distinct possibility.

A second hypothesis for the observed elimination of Zn2+ antagonism in the K260H/H231A/E233A/E238A mutant is that the triple alanine mutation causes structural changes in the lip of the ATD lobe, thereby disrupting a Zn2+ site between the introduced histidine and ligands elsewhere in the receptor. However, a couple of observations are not in line with this hypothesis. First, all of the mutants where single or double alanine mutations have been introduced in K260H display pharmacological profiles of Zn2+ not significantly different from that of K260H (Table I). It seems unlikely that this indirect disruption of the Zn2+ antagonism by the triple alanine mutation would not be seen in any of the mutants containing double alanine mutations. Second, the antagonistic effect of zinc is retained when the three histidine/glutamate coligands are mutated to glutamates and histidines, respectively (mutant K260H/H231E/E233H/E238H). In many ways, this combination of glutamate/histidine mutations represents a much more drastic electronic and structural alteration of the receptor protein than the triple alanine mutation in K260H/H231A/E233A/E238A. Furthermore, the increased antagonistic potency of Zn2+ on mutant K260H/H231E/E233H/E238H compared with K260H strongly supports the proposal of the involvement of one or more of the three coligands in zinc binding.

While only one of the three endogenous coligands had to be present in the mGluR1 ATD for Zn2+ to exert its effect, the histidine introduced instead of Lys260 appeared to be crucial for Zn2+ binding. No antagonistic effect of Zn2+ was observed on the WT receptor or mutant K260A, most likely because the metal ion does not bind to these receptors. Alternatively, zinc may bind "silently" to the three endogenous "coligands" in WT and K260A. In both scenarios, Zn2+ binding clearly has to involve residues on two strands in the ATD lip to have any implications for the signal transduction through the receptor. Analogously, both antagonistic and agonistic zinc sites created by mutations in one TM of family A and B GPCRs have been significantly "improved" by the introduction of coordinating zinc ligands in another TM (39, 42-44, 46).

The fact that mGluR1 activation can be inhibited by Zn2+ binding to two strands in the ATD lip not involved in agonist binding is very interesting. Binding of the metal ion to K260H mGluR1 does not appear to disrupt the dimerization of the receptor (Fig. 7). The possibility that binding of the metal ion could cause a "twist" in the orientation between the two receptors in the homodimer interface, hereby affecting receptor functionality, cannot be completely excluded. However, we favor an explanation where the effects of Zn2+ arise solely from intramolecular structural changes in the receptor protein. We propose that Zn2+ binding pins the two strands in the lip of the ATD lobe together and introduces a conformational "lock" on the region. The Zn2+-bound lobe is incapable of participating in the contraction of the ATD, which becomes trapped in a rigid, inactivated conformation. Alternately, Zn2+ binding to the ATD lip of K260H could also be speculated to interfere with the intramolecular communication between the ATD and the seven-transmembrane moiety (7TM) of the family C receptor. We and others have suggested a mechanism underlying the signal transduction through the receptor, where the signal is transferred to the G-protein coupling area by an intramolecular interaction between the ATD and the 7TM of the receptor (1, 9, 53). Hence, the antagonistic effect of Zn2+ binding could be thought to arise from a disruption of a structural motif in the ATD lobe necessary for the signal transfer process.

In contrast to the antagonistic effect of Zn2+ on K260H mGluR1, Cu2+ and Co2+ displayed no measurable inhibition of the agonist-induced response in the nontoxic concentration range of the metal ions. This indicates that the site constructed in K260H is relatively specific for Zn2+. The effect of Cu2+ (as the free ion or complexed with phenanthroline) on metal ion sites constructed in TMs of family A GPCRs have varied from being just as potent to being significantly weaker than that of that of Zn2+ (41, 42, 44). Whether Cu2+ and Co2+ do not bind to K260H or whether they bind but, unlike Zn2+, are unable to induce a conformational lock on the region is impossible to determine.

It is quite interesting that introduction of a structural constraint in the ATD lobe opposite to the "agonist-binding" lobe of mGluR1 has such pronounced consequences for receptor functionality. The local flexibility in the ATD lip, which may enable Zn2+ to coordinate to the introduced histidine and three different endogenous coligands is contrasted by the apparent small degree of overall structural flexibility of the ATD region, resulting in its sensitivity toward "locally applied rigidity." Hence, the zinc site created in mGluR1 is quite interesting from a medicinal chemistry point of view. From a therapeutic perspective the interest in the field of mGluR ligands has classically been focused on antagonists for the group I mGluRs and agonists for the group II and III mGluRs (1, 9, 10). The vast majority of antagonists for mGluRs reported so far have been competitive antagonists constructed from the amino acid structure of the endogenous agonist (1, 10). However, in recent years a new generation of noncompetitive antagonists has been introduced (54-58), of which CPCCOEt and 2-methyl-6-(phenylethynyl)pyridine have been demonstrated to bind to the 7TM of mGluR1 and mGluR5, respectively (23, 53, 59). The present study is the first report of noncompetitive antagonism originating from ligand binding to a region in the ATD. The "functional antagonism" exerted by Zn2+ on the ATD of K260H mGluR1 in this study resembles the actions of CPCCOEt on mGluR1 in some respects. CPCCOEt binds to the top of TM7 in mGluR1, and it has been speculated that it in this way interferes with the signaling between the ATD and the 7TM (53). Analogously, Zn2+ also blocks the signal transduction through the receptor, either through a similar interference in the ATD/7TM communication or by blocking ATD contraction a step prior to this "intramolecular cross-talk." We propose that the lips of the two globular domains of the ATD could be very interesting targets in the design of new antagonists for mGluR1 and the other mGluRs. Furthermore, certain compounds interacting with the lips of the two ATD lobes could possibly also function as allosteric modulators of mGluR activation. In that respect, the increased basal response achieved by a Glu238 right-arrow His mutation in mGluR1b is quite interesting (Fig. 6). Allosteric activators of another family C GPCR, CaR, have been reported, but the binding sites of these compounds have not been disclosed (60, 61). Ligands directed toward the ATD lips of the family C GPCRs could be very different structurally from that of agonists and competitive antagonists, which again could be a way of overcoming traditional drug delivery obstacles such as metabolism and low blood-brain barrier penetration and, in the case of mGluR and GABAB ligands, reduce the risk of the compound being taken up by glutamate and GABA transporters.

In conclusion, this study represents the first zinc site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. As it was to be expected from the existence of numerous endogenous metal ion binding sites in proteins (27-29, 48), the use of zinc as a structural probe in GPCRs can clearly be extended to regions outside of the "confinement" of the membrane. While not all ligands for Zn2+ in K260H mGluR1 may have been identified, the findings in this study offer valuable insight into the mechanism of ATD closure and family C receptor activation, identify regions in the ATD as interesting targets from a medicinal chemistry point of view, and strongly support our previously published model of the ATD of mGluR1 (11).

    ACKNOWLEDGEMENTS

We thank Professor Shigetada Nakanishi, Professor Solomon H. Snyder, and Dr. Mark R. Brann for generous gifts of cDNAs encoding mGluR1a, CaR, and m5, respectively. Dr. Penelope S. V. Jones and Professor Nakanishi are also thanked for generous gifts of the tsA and mGluR1a-CHO cell lines, respectively. The kind guidance of Dr. Carl Romano with respect to the design of the Western blot experiments is greatly appreciated.

    Note Added in Proof

The recently published crystal structures of the mGluR1 ATD homodimer (Kunishima et al. (2000) Nature 407, 971-977) have shedded light on the created zinc site in mGluR1b. According to the crystal structures, the introduced histidine in K260H mGluR1b and the three proposed coligands for Zn2+, His231, Glu233, and Glu238 are located in close proximity in both of the ATDs in the dimer. Whereas the two Zn2+ sites in the two ATDs are located far apart in the "resting," inactive dimer conformation, they are facing each other across the cleft between the ATDs in the "active"dimer conformation. Hence, we propose that Zn2+ binding to one or both of the zinc sites prevents the conformational transition of the ATD dimer from the "resting" to the "active" state due to steric interactions.

    FOOTNOTES

* This work was supported by grants from the Danish Medical Research Council and the Novo Nordisk Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. Tel.: 45-3530-6518; Fax: 45-3530-6040; E-mail: hbo@dfh.dk.

Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007220200

2 L. Prézeau and J.-P. Pin, personal communication.

    ABBREVIATIONS

The abbreviations used are: Glu, (S)-glutamic acid; mGluR, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; GABA, gamma -aminobutyric acid; GABABR, gamma -aminobutyric acid receptor type B; CaR, calcium-sensing receptor; ATD, amino-terminal domain; PBP, periplasmic binding protein; [3H]Quis, [3H]quisqualic acid; (S)-4CPG, (S)-4-Carboxyphenylglycine; CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylic acid ethyl ester; IP, inositol phosphate; HBSS, Hanks' balanced saline solution; WT, wild type; PBS, phosphate-buffered saline solution; CHO, Chinese hamster ovary; TM, transmembrane region; 7TM, seven-transmembrane moiety.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Bräuner-Osborne, H., Egebjerg, J., Nielsen, E. Ø., Madsen, U., and Krogsgaard-Larsen, P. (2000) J. Med. Chem. 43, 2609-2645[CrossRef][Medline] [Order article via Infotrieve]
2. Möhler, H., and Fritschy, J.-M. (1999) Trends Pharmacol. Sci. 20, 87-89[CrossRef][Medline] [Order article via Infotrieve]
3. Brown, E. M. (1999) Am. J. Med. 106, 238-253[CrossRef][Medline] [Order article via Infotrieve]
4. Tirindelli, R., Mucignat-Caretta, C., and Ryba, N. J. (1999) Trends Neurosci. 21, 482-486[CrossRef]
5. Cheng, V., and Lotan, R. (1998) J. Biol. Chem. 273, 35008-35015[Abstract/Free Full Text]
6. Bräuner-Osborne, H., and Krogsgaard-Larsen, P. (2000) Genomics 65, 121-128[CrossRef][Medline] [Order article via Infotrieve]
7. Robbins, M. J., Michalovich, D., Hill, J., Calver, A. R., Medhurst, A. D., Gloger, I., Sims, M., Middlemiss, D. N., and Pangalos, M. N. (2000) Genomics 67, 8-18[CrossRef][Medline] [Order article via Infotrieve]
8. Nakanishi, S. (1994) Neuron 13, 1031-1037[Medline] [Order article via Infotrieve]
9. Pin, J-P., de Colle, C., Bessis, A. S., and Acher, F. (1999) Eur. J. Pharmacol. 375, 277-294[CrossRef][Medline] [Order article via Infotrieve]
10. Schoepp, D. D., Jane, D. E., and Monn, J. A. (1999) Neuropharmacology 38, 1431-1476[CrossRef][Medline] [Order article via Infotrieve]
11. O'Hara, P. J., Sheppard, P. O., Thøgersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. H., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) Neuron 11, 41-52[Medline] [Order article via Infotrieve]
12. Takahishi, K., Tsuchida, K., Tanabe, Y., Masayuki, M., and Nakanishi, S. (1993) J. Biol. Chem. 268, 19341-19345[Abstract/Free Full Text]
13. Tones, M. A., Bendali, N., Flor, P. J., Knöpfel, T., and Kuhn, R. (1995) Neuroreport 7, 117-120[Medline] [Order article via Infotrieve]
14. 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]
15. Han, G., and Hampson, D. R. (1999) J. Biol. Chem. 274, 10008-10013[Abstract/Free Full Text]
16. Bräuner-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]
17. Malitschek, B., Schweizer, C., Keir, M., Heid, J., Froestl, W., Mosbacher, J., Kuhn, R., Henley, J., Joly, C., Pin, J.-P., Kaupmann, K., and Bettler, B. (1999) Mol. Pharmacol. 56, 448-454[Abstract/Free Full Text]
18. Hampson, D. R., Huang, X-P., Pekhletski, R., Peltekova, V., Hornby, G., Thomsen, C., and Thøgersen, H. (1999) J. Biol. Chem. 274, 33488-33495[Abstract/Free Full Text]
19. Galvez, T., Parmentier, M-L., Joly, C., Malitschek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., and Pin, J-P. (1999) J. Biol. Chem. 274, 13362-13369[Abstract/Free Full Text]
20. Ray, K., Hauschild, B. C., Steinbach, P. J., Goldsmith, P. K., Hauache, O., and Spiegel, A. M. (1999) J. Biol. Chem. 274, 27642-27650[Abstract/Free Full Text]
21. Quiocho, F. A., and Ledvina, P. S. (1996) Mol. Microbiol. 20, 17-25[Medline] [Order article via Infotrieve]
22. Jensen, A. A., Sheppard, P. O., O'Hara, P. J., Krogsgaard-Larsen, P., and Bräuner-Osborne, H. (2000) Eur. J. Pharmacol. 397, 247-253[CrossRef][Medline] [Order article via Infotrieve]
23. Bräuner-Osborne, H., Jensen, A. A., and Krogsgaard-Larsen, P. (1999) Neuroreport 10, 3923-3925[Medline] [Order article via Infotrieve]
24. Chahine, M., Bennett, P. B., George, A. L., Jr., and Horn, R. (1994) Pflügers Arch. 427, 136-142[Medline] [Order article via Infotrieve]
25. Nanevicz, T., Wang, L., Chen, M., Ishii, M., and Coughlin, S. R. (1996) J. Biol. Chem. 271, 702-706[Abstract/Free Full Text]
26. Jensen, A. A., Spalding, T. A., Burstein, E. S., Sheppard, P. O., O'Hara, P. J., Brann, M. R., Krogsgaard-Larsen, P., and Bräuner-Osborne, H. (2000) J. Biol. Chem. 275, 29547-29555[Abstract/Free Full Text]
27. Christianson, D. W. (1991) Adv. Protein Chem. 42, 281-355[Medline] [Order article via Infotrieve]
28. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281[CrossRef][Medline] [Order article via Infotrieve]
29. Alberts, I. L., Nadassy, K., and Wodak, S. J. (1998) Protein Sci. 7, 1700-1716[Abstract/Free Full Text]
30. Loland, C. J., Nørregaard, L., and Gether, U. (1999) J. Biol. Chem. 274, 36928-36934[Abstract/Free Full Text]
31. Romano, C., Yang, W. -L., and O'Malley, K. L. (1996) J. Biol. Chem. 271, 28612-28616[Abstract/Free Full Text]
32. Romano, C., Miller, J., Hyre, K., Mennerick, S., Dikranian, S., Takeuchi, Y., and O'Malley, K. L. (1999) Neuropharmacology 38, 37[CrossRef] (abstr.)
33. Tsuji, Y., Shimada, Y., Takeshita, T., Kajimura, N., Nomura, S., Sekiyama, N., Otomo, J., Usukura, J., Nakanishi, S., and Jingami, H. (2000) J. Biol. Chem. 275, 28144-28151[Abstract/Free Full Text]
34. Reid, S. N., Romano, C., Hughes, T., and Daw, N. W. (1995) J. Comp. Neurol. 355, 470-477[Medline] [Order article via Infotrieve]
35. Doherty, A. J., Coutinho, V., Collingridge, G. L., and Henley, J. M. (1999) Biochem. J. 341, 415-422[CrossRef][Medline] [Order article via Infotrieve]
36. Ray, K., and Hauschild, B. C. (2000) J. Biol. Chem. 275, 34245-34251[Abstract/Free Full Text]
37. Pace, A. J., Gama, L., and Breitwieser, G. E. (1999) J. Biol. Chem. 274, 11629-11634[Abstract/Free Full Text]
38. Galvez, T., Prézeau, 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]
39. Elling, C. E., Nielsen, S. M., and Schwartz, T. W. (1995) Nature 374, 74-77[CrossRef][Medline] [Order article via Infotrieve]
40. Elling, C. E., Thirstrup, K., Nielsen, S. M., Hjorth, S. A., and Schwartz, T. W. (1997) Fold. Des. 2, S76-S80[Medline] [Order article via Infotrieve]
41. Elling, C. E., Thirstrup, K., Nielsen, S. M., Hjorth, S. A., and Schwartz, T. W. (1997) Ann. N. Y. Acad. Sci. 814, 142-151[Medline] [Order article via Infotrieve]
42. Elling, C. E., Thirstrup, K., Holst, B., and Schwartz, T. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12322-12327[Abstract/Free Full Text]
43. Sheikh, S. P., Vilardarga, J. P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041[Abstract/Free Full Text]
44. Holst, B., Elling, C. E., and Schwartz, T. W. (2000) Mol. Pharmacol. 58, 263-270[Abstract/Free Full Text]
45. Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350[CrossRef][Medline] [Order article via Infotrieve]
46. Lu, Z-L., and Hulme, E. C. (2000) J. Biol. Chem. 275, 5682-5686[Abstract/Free Full Text]
47. Chakrabarti, P. (1990) Protein Eng. 4, 57-63[Abstract]
48. Kiefer, L. L., and Fierke, C. A. (1994) Biochemistry 33, 15233-15240[Medline] [Order article via Infotrieve]
49. Lesburg, C. A., Huang, C., Christianson, D. W., and Fierke, C. A. (1997) Biochemistry 36, 15780-15791[CrossRef][Medline] [Order article via Infotrieve]
50. Regan, L. (1995) Trends Biochem. Sci. 20, 280-285[CrossRef][Medline] [Order article via Infotrieve]
51. McGrath, M. E., Haymore, B. L., Summers, N. L., Craik, C. S., and Fletterick, R. J. (1993) Biochemistry 32, 1914-1919[Medline] [Order article via Infotrieve]
52. Ippolito, J. A., and Christianson, D. W. (1994) Biochemistry 33, 15241-15249[Medline] [Order article via Infotrieve]
53. Litschig, S., Gasparini, F., Rueegg, D., Stoehr, N., Flor, P. J., Vranesic, I., Prezeau, L., Pin, J-P., Thomsen, C., and Kuhn, R. (1999) Mol. Pharmacol. 55, 453-461[Abstract/Free Full Text]
54. Annoura, H., Fukunaga, A., and Uesugi, M. (1996) Bioorg. Med. Chem. Lett. 6, 763-766[CrossRef]
55. Varney, M. A., Cosford, N. D., Jachec, C., Rao, S. P., Sacaan, A., Lin, F. F., Bleicher, L., Santori, E. M., Flor, P. J., Allgeier, H., Gasparini, F., Kuhn, R., Hess, S. D., Velicelebi, G., and Johnson, E. C. (1999) J. Pharmacol. Exp. Ther. 290, 170-181[Abstract/Free Full Text]
56. Gasparini, F., Lingenhöhl, K., Stoehr, N., Flor, P. J., Heinrich, M., Vranesic, I., Biollaz, M., Allgeier, H., Heckendorn, R., Urwyler, S., Varney, M. A., Johnson, E. C., Hess, S. D., Rao, S. P., Sacaan, A. I., Santori, E. M., Velicelebi, G., and Kuhn, R. (1999) Neuropharmacology 38, 1493-1503[CrossRef][Medline] [Order article via Infotrieve]
57. Kolczewski, S., Adam, G., Stadler, H., Mutel, V., Wichmann, J., and Woltering, T. (1999) Bioorg. Med. Chem. 9, 2173-2176[CrossRef]
58. Wichmann, J., Adam, G., Kolczewski, S., Mutel, V., and Woltering, T. (1999) Bioorg. Med. Chem. 9, 1573-1576[CrossRef]
59. Pagano, A., Rüegg, D., Litschig, S., Stoehr, N., Stierlin, C., Heinrich, M., Floersheim, P., Prezèau, L., Carroll, F., Pin, J-P., Cambria, A., Vranesic, I., Flor, P. J., Gasparini, F., and Kuhn, R. (2000) J. Biol. Chem. 275, 33750-33758[Abstract/Free Full Text]
60. Nemeth, E. F., Steffey, M. E., Hammerland, L. G., Hung, B. C. P., van Wagenen, B. C., DelMar, E. G., and Balandrin, M. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4040-4045[Abstract/Free Full Text]
61. Hammerland, L. G., Garrett, J. E., Hung, B. C. P., Levinthal, C., and Nemeth, E. F. (1998) Mol. Pharmacol. 53, 1083-1088[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.