The parasagittal distribution of the splice variant 1b of the metabotropic glutamate receptor 1 (... more The parasagittal distribution of the splice variant 1b of the metabotropic glutamate receptor 1 (mGluR1b) was studied in the cerebellar cortex by using a specific mGluR1b antiserum combined with immunocytochemical methods. In f rontal cerebellar sections, the antibodies revealed alternating bands of immunopositive and immunonegative Purkinje cells in lobules I to X of the vermis. In these regions, the distribution of mGluR1b was complementary to longitudinal bands of Purkinje cells expressing Zebrin II. The width of the mGluR1b-positive bands d e c reased pro g ressively in the ro s t ro-c a u d a l direction, contrary to the rostro-caudal increase in the width of the Zebrin-positive bands. In the hemispheres, mGluR1b-positive bands alternated with Zebrin II-immunopositive bands in the paravermal portions of Crus I and Crus II. MGluR1b and Zebrin II immunoreactivities often colocalized in other regions of the hemispheral lobules. The compartmentalization of mGluR1b suggests that adjacent Purkinje cells might respond d i ff e rently depending on the presence of mGluR1b at parallel fiber-Purkinje cell synapses.
L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is ... more L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is mediated via ionotropic and metabotropic glutamate receptors (mGluR). Eight mGluRs have already been cloned. In the present study the hypothalamic distribution of mGluR1a has been investigated by immunocytochemistry using monoclonal antibodies recently produced by some of the present authors (T. J. G., R. K., T. K.). The observations have been compared with findings obtained with a polyclonal antibody. A widespread and heterogeneous distribution of mGluR1a was found with the monoclonal antibodies. Intense immunolabelling of perikarya and dendrites occurred in several hypothalamic cell groups including the suprachiasmatic, anterior periventricular, anterior hypothalamic (posterior part), paraventricular, supraoptic, arcuate, tuberal magnocellular, dorsomedial and mammillary nuclei (particularly in the medial). It was only the ventromedial nucleus in which several perikarya were stained by the polyclonal antibody but appeared to be negative by the monoclonal antibodies. The findings fit extremely well with the data on the hypothalamic distribution of mGluR1 mRNA with the exception of the ventromedial nucleus. It remains to be elucidated whether alternatively spliced variants of mGluR1 (mGluR1b and 1c) are expressed in this nucleus. Further, they confirm the results of former immunohistochemical studies. In addition, they indicate that a significant part of the neuroendocrine region of the hypothalamus (including the paraventricular, supraoptic and arcuate nuclei) also contains mGluR1 suggesting that this receptor may play a role also in neuroendocrine regulation.
It has been proposed that neurotransmitter signalling can occur between axons and glia in the mam... more It has been proposed that neurotransmitter signalling can occur between axons and glia in the mammalian optic nerve in the absence of synaptic specialisations, and that this may be glutamate mediated. Here, the cellular distribution of five metabotropic glutamate receptors (mGluR's 1a, 1b, 1c, 2/3 and 5) have been assessed in the rat optic pathway using specific antibodies. Positive immunoreactivity is found for mGluR2/3 and 5. Both are found in axons, although only mGluR5 is present in the majority of these. Strong immunoreactivity for mGluR2/3 is found in cells in the optic pathway and thalamus. The cellular morphology and distribution is consistent with their being astrocytes. Examination of brain sections stained for mGluR2/3 is consistent with this notion, with many cells having end-feet processes terminating on blood vessels or the pial surface. The axonal immunoreactivity could represent the presence of these receptors on axons, but it is more probable that the receptor protein synthesised in the ganglion cell soma is being transported to the cell terminal in sufficient concentration to be revealed by immunohistochemistry. The reason for the axon-astrocyte signalling is unclear, and may be associated with metabolic coupling. In development, communication between axons and glia mediates a range of functions including pathway selection and myelination. It is probable that in the adult this form of signalling underpins a range of functions that have yet to be described.
A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from huma... more A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from human brain cDNA libraries by cross‐hybridization with rat mGluR2 probes. The deduced amino acid sequence of the human mGluR2 receptor consists of 872 residues and shows a sequence identity of 97% to the amino acid sequence of rat mGluR2. Northern blot analyses showed that hmGluR2 is widely expressed in different regions of the adult brain as well as in fetal human brain. Genomic Southern blotting localized the mGluR2 gene to human chromosome 3. Chinese hamster ovary (CHO) cells stably transfected with the cloned hmGluR2 cDNA exhibit agonist induced depression of forskolin‐stimulated cAMP accumulation. A direct comparison of CHO cells stably expressing human and rat mGluR2 with five agonists revealed the same rank order of potency [(2S,3S,4S)‐α‐(carboxycyclopropyl)‐glycine » (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid =l‐glutamate » quisqualate =l‐2‐amino‐4‐phosphonobutyric acid] and similar EC50 values for both homologous receptors. (R.S)‐a‐methyl‐4‐carboxyphenylglycine, a reported antagonist at some mGluR subtypes, reduced the depression of forskolin‐induced cAMP accumulation by (1S,3R)‐ACPD in both human and rat mGluR2.
Proceedings of the National Academy of Sciences of the United States of America, Sep 27, 2018
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CA... more Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the gene (). Although mutant HTT is expressed during embryonic development and throughout life, clinical HD usually manifests later in adulthood. A number of studies document neurodevelopmental changes associated with mutant , but whether these are reversible under therapy remains unclear. Here, we identify very early behavioral, molecular, and cellular changes in preweaning transgenic HD rats and mice. Reduced ultrasonic vocalization, loss of prepulse inhibition, and increased risk taking are accompanied by disturbances of dopaminergic regulation in vivo, reduced neuronal differentiation capacity in subventricular zone stem/progenitor cells, and impaired neuronal and oligodendrocyte differentiation of mouse embryo-derived neural stem cells in vitro. Interventional treatment of this early phenotype with the histone deacetylase inhibitor (HDACi) LBH589 led to significant impr...
The Journal of pharmacology and experimental therapeutics, 1999
Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of exci... more Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobuty...
Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded ... more Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.
The ␥-aminobutyric acid type B (GABA B) receptor is distantly related to the metabotropic glutama... more The ␥-aminobutyric acid type B (GABA B) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABA B receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteinerich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABA B receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABA B receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABA B receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.
We have investigated the mechanism of inhibition and site of action of the novel human metabotrop... more We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [ 3 H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [ 3 H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPC-COEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.
The cDNA encoding hmGluR6, appended with a 15amino acid antibody epitope (1D4), was transiently t... more The cDNA encoding hmGluR6, appended with a 15amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the ␣ subunit of G o was assayed in vitro using a guanosine 5-3-O-(thio)triphosphate binding assay. Activation of both transducin and G o was observed. The rate of G o activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to G o is more efficient and suggests that G o may be involved in coupling to mGluR6 in ON-bipolar cells.
The parasagittal distribution of the splice variant 1b of the metabotropic glutamate receptor 1 (... more The parasagittal distribution of the splice variant 1b of the metabotropic glutamate receptor 1 (mGluR1b) was studied in the cerebellar cortex by using a specific mGluR1b antiserum combined with immunocytochemical methods. In f rontal cerebellar sections, the antibodies revealed alternating bands of immunopositive and immunonegative Purkinje cells in lobules I to X of the vermis. In these regions, the distribution of mGluR1b was complementary to longitudinal bands of Purkinje cells expressing Zebrin II. The width of the mGluR1b-positive bands d e c reased pro g ressively in the ro s t ro-c a u d a l direction, contrary to the rostro-caudal increase in the width of the Zebrin-positive bands. In the hemispheres, mGluR1b-positive bands alternated with Zebrin II-immunopositive bands in the paravermal portions of Crus I and Crus II. MGluR1b and Zebrin II immunoreactivities often colocalized in other regions of the hemispheral lobules. The compartmentalization of mGluR1b suggests that adjacent Purkinje cells might respond d i ff e rently depending on the presence of mGluR1b at parallel fiber-Purkinje cell synapses.
L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is ... more L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is mediated via ionotropic and metabotropic glutamate receptors (mGluR). Eight mGluRs have already been cloned. In the present study the hypothalamic distribution of mGluR1a has been investigated by immunocytochemistry using monoclonal antibodies recently produced by some of the present authors (T. J. G., R. K., T. K.). The observations have been compared with findings obtained with a polyclonal antibody. A widespread and heterogeneous distribution of mGluR1a was found with the monoclonal antibodies. Intense immunolabelling of perikarya and dendrites occurred in several hypothalamic cell groups including the suprachiasmatic, anterior periventricular, anterior hypothalamic (posterior part), paraventricular, supraoptic, arcuate, tuberal magnocellular, dorsomedial and mammillary nuclei (particularly in the medial). It was only the ventromedial nucleus in which several perikarya were stained by the polyclonal antibody but appeared to be negative by the monoclonal antibodies. The findings fit extremely well with the data on the hypothalamic distribution of mGluR1 mRNA with the exception of the ventromedial nucleus. It remains to be elucidated whether alternatively spliced variants of mGluR1 (mGluR1b and 1c) are expressed in this nucleus. Further, they confirm the results of former immunohistochemical studies. In addition, they indicate that a significant part of the neuroendocrine region of the hypothalamus (including the paraventricular, supraoptic and arcuate nuclei) also contains mGluR1 suggesting that this receptor may play a role also in neuroendocrine regulation.
It has been proposed that neurotransmitter signalling can occur between axons and glia in the mam... more It has been proposed that neurotransmitter signalling can occur between axons and glia in the mammalian optic nerve in the absence of synaptic specialisations, and that this may be glutamate mediated. Here, the cellular distribution of five metabotropic glutamate receptors (mGluR's 1a, 1b, 1c, 2/3 and 5) have been assessed in the rat optic pathway using specific antibodies. Positive immunoreactivity is found for mGluR2/3 and 5. Both are found in axons, although only mGluR5 is present in the majority of these. Strong immunoreactivity for mGluR2/3 is found in cells in the optic pathway and thalamus. The cellular morphology and distribution is consistent with their being astrocytes. Examination of brain sections stained for mGluR2/3 is consistent with this notion, with many cells having end-feet processes terminating on blood vessels or the pial surface. The axonal immunoreactivity could represent the presence of these receptors on axons, but it is more probable that the receptor protein synthesised in the ganglion cell soma is being transported to the cell terminal in sufficient concentration to be revealed by immunohistochemistry. The reason for the axon-astrocyte signalling is unclear, and may be associated with metabolic coupling. In development, communication between axons and glia mediates a range of functions including pathway selection and myelination. It is probable that in the adult this form of signalling underpins a range of functions that have yet to be described.
A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from huma... more A cDNA encoding the human metabotropic glutamate receptor type 2 (hmGluR2) was isolated from human brain cDNA libraries by cross‐hybridization with rat mGluR2 probes. The deduced amino acid sequence of the human mGluR2 receptor consists of 872 residues and shows a sequence identity of 97% to the amino acid sequence of rat mGluR2. Northern blot analyses showed that hmGluR2 is widely expressed in different regions of the adult brain as well as in fetal human brain. Genomic Southern blotting localized the mGluR2 gene to human chromosome 3. Chinese hamster ovary (CHO) cells stably transfected with the cloned hmGluR2 cDNA exhibit agonist induced depression of forskolin‐stimulated cAMP accumulation. A direct comparison of CHO cells stably expressing human and rat mGluR2 with five agonists revealed the same rank order of potency [(2S,3S,4S)‐α‐(carboxycyclopropyl)‐glycine » (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid =l‐glutamate » quisqualate =l‐2‐amino‐4‐phosphonobutyric acid] and similar EC50 values for both homologous receptors. (R.S)‐a‐methyl‐4‐carboxyphenylglycine, a reported antagonist at some mGluR subtypes, reduced the depression of forskolin‐induced cAMP accumulation by (1S,3R)‐ACPD in both human and rat mGluR2.
Proceedings of the National Academy of Sciences of the United States of America, Sep 27, 2018
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CA... more Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded CAG repeats in the gene (). Although mutant HTT is expressed during embryonic development and throughout life, clinical HD usually manifests later in adulthood. A number of studies document neurodevelopmental changes associated with mutant , but whether these are reversible under therapy remains unclear. Here, we identify very early behavioral, molecular, and cellular changes in preweaning transgenic HD rats and mice. Reduced ultrasonic vocalization, loss of prepulse inhibition, and increased risk taking are accompanied by disturbances of dopaminergic regulation in vivo, reduced neuronal differentiation capacity in subventricular zone stem/progenitor cells, and impaired neuronal and oligodendrocyte differentiation of mouse embryo-derived neural stem cells in vitro. Interventional treatment of this early phenotype with the histone deacetylase inhibitor (HDACi) LBH589 led to significant impr...
The Journal of pharmacology and experimental therapeutics, 1999
Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of exci... more Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobuty...
Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded ... more Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.
The ␥-aminobutyric acid type B (GABA B) receptor is distantly related to the metabotropic glutama... more The ␥-aminobutyric acid type B (GABA B) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABA B receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteinerich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABA B receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABA B receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABA B receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.
We have investigated the mechanism of inhibition and site of action of the novel human metabotrop... more We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [ 3 H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [ 3 H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPC-COEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.
The cDNA encoding hmGluR6, appended with a 15amino acid antibody epitope (1D4), was transiently t... more The cDNA encoding hmGluR6, appended with a 15amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the ␣ subunit of G o was assayed in vitro using a guanosine 5-3-O-(thio)triphosphate binding assay. Activation of both transducin and G o was observed. The rate of G o activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to G o is more efficient and suggests that G o may be involved in coupling to mGluR6 in ON-bipolar cells.
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Papers by Rainer Kuhn