Group III human metabotropic glutamate receptors 4, 7 and 8: Molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells

doi:10.1016/S0169-328X(97)00277-5

Molecular Brain Research

Volume 53, Issues 1–2, January 1998, Pages 88-97

https://doi.org/10.1016/S0169-328X(97)00277-5 Get rights and content

Abstract

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67–70% amino acid similarity with each other and 42–45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist l-AP4 varied greatly between the group III clones, being mGluR8>mGluR4≫mGluR7. The reported group II mGluR agonist l-CCG-I was a highly potent mGluR8 agonist (EC₅₀=0.35 μM), with significant agonist activities at both mGluR4 (EC₅₀=3.7 μM) and mGluR7 (EC₅₀=47 μM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8≫mGluR4=mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.

Introduction

l-Glutamate is a major excitatory neurotransmitter which plays an important role in many central neuronal functions and dysfunctions such as memory acquisition, learning, epilepsy and stroke as well as some neurodegenerative disorders (For reviews, see 1, 2, 3, 4, 5). This neurotransmitter interacts with two distinct types of receptors namely ionotropic and metabotropic glutamate receptors. The ionotropic glutamate (iGlu) receptors contain integral cation specific ion channels and are subdivided into three groups namely N-methyl-d-asparate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate (KA), on the basis of pharmacological and electrophysiological studies 2, 6, 7, 4, 8. In contrast, the metabotropic glutamate (mGlu) receptors represent a novel class of G-protein coupled receptors which are both functionally and pharmacologically different from the iGlu receptor family 9, 10.

Molecular cloning studies have identified eight rat mGluR subtypes which can be divided into group I, group II and group III on the basis of sequence homology, putative signal transduction mechanisms and pharmacological properties 11, 12, 13, 14, 15, 16, 17, 18, 19, 9, 20, 21, 22, 23, 8, 10, 24, 25. Group I includes mGluR1 and mGluR5 which are coupled to phospholipase C (PLC) and are selectively activated by 3,5-dihydroxyphenyl glycine (DHPG) at low μM concentrations. In contrast, group II receptors (including mGluR2 and mGluR3) as well as group III receptors (mGluR4, mGluR6, mGluR7 and mGluR8) are all negatively coupled to adenylate cyclase, thereby depressing elevations in cAMP levels.

Agonist selectivity has also been useful to differentiate between groups II and III mGluRs. Prototypic group II mGluR agonists include (2S,3S,4S)-α-(carboxy cyclopropyl) glycine (l-CCG I) [26], (2S,1ÕR,2ÕR,3ÕR)-2-(2Õ,3Õ-dicarboxy cyclopropyl) glycine (DCG-IV) [27], 2R,4R-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) 28, 29, and LY354740 30, 31. Group III agonists include l-2-amino-4-phosphonobutyrate (l-AP4) and l-serine-O-phosphate (l-SOP) [22]. However, the known subtype selectivities of these mGluR agonists are confounded by the lack of data comparing each mGluR in the same cell system against all receptors within that group. This is related in part to the weak coupling of certain group III mGluRs to second messenger systems in some non-neuronal cells. For example, activation of human mGluR4 receptors [13]in CHO cells by the group III agonist l-AP4 has been shown to produce about 50% maximal inhibition of forskolin-stimulated cAMP formation. When mouse mGluR8 was expressed in the same cells, the maximal effect of l-AP4 was only about 10% inhibition of forskolin-stimulated cAMP [12], making it difficult to perform and interpret their pharmacological studies. Since mGluR8 apparently coupled weakly to inhibition of cAMP formation, Saugstad et al. [32]resorted to evoking potassium currents in Xenopus oocytes by co-expressing both rat mGluR8 and G-protein coupled inwardly rectifying potassium channels as a means to study the pharmacological properties of this receptor.

In this study, we report the cloning, expression, and pharmacological properties of the human versions of group III mGluRs 4, 7, and 8. These receptors were expressed in `RGT' cells. RGT cells are AV12-664 cells co-expressing a glutamate–aspartate transporter protein [33]. We have shown previously that this cell line can be used to study the pharmacological properties of other mGluRs including the phosphoinositide coupled receptors human mGluR1a and mGluR5a 34, 35, and group II human mGluRs 2 and 3 29, 31which couple to inhibition of forskolin-stimulated cAMP formation. Using this cell system, robust coupling to inhibition of cAMP formation was observed for each group III mGluR, including human mGluR4, 7, and 8. Thus, we report here cloning and pharmacological comparisons of human mGluR4, 7, and 8, with each clone expressed in the same (RGT) cell system. It should be noted that Flor et al. [13]and Makoff et al. 17, 18recently described the cloning, expression and pharmacology of human mGluR4 and mGluR7 in CHO cells.

Section snippets

Materials

Materials were obtained from the following sources: 1S,3R-ACPD, l-AP4, l-CCG I, DHPG, MPPG, MCPG, NMDA, AMPA, kainate, and l-SOP were obtained from Tocris Cookson (St. Louis, MO). 2R,4R-APDC was synthesized as described by Monn et al. [36]and LY354740 was synthesized as described by Monn et al. [30]. All molecular biology reagents and enzymes were purchased from either Life Technologies (Gaithersburg, MD), Boehringer Mannheim Corporation (Indianapolis, IN), New England Biolabs (Beverley, MA),

Results

Full length cDNA clones encoding mGluR4, mGluR7 and mGluR8 were isolated as described in Section 2.2(Fig. 1). These cDNA inserts were modified at their 5′ and 3′ ends to contain either SalI or SalI/KpnI restriction sites and then digested with either SalI or SalI/KpnI enzymes to generate 3063, 3018 and 3400 bp restriction fragments containing coding sequences for mGluR4, mGluR7 and mGluR8, respectively. To determine the functional properties of the cloned mGluR4, mGluR7 and mGluR8 receptors,

Discussion

In this paper, the cloning, characterization, functional expression in RGT cells and subsequent pharmacological properties of three members of group III human metabotropic glutamate receptors (mGluR4, mGluR7 and mGluR8) were described. The sequences of these cloned human receptors (Gene Bank accession numbers U92457, U92458 and U92459) were highly similar to the rat mGluR4, mGluR7 and mouse mGluR8 which were reported previously 12, 21, 10, 24. The cDNA sequences for human mGluR4, mGluR7 and

Acknowledgements

We would like to thank Dr. J. Paul Burnett and Nancy G. Mayne for providing the RGT cell line, helpful discussions and suggestions. We thank Bruce Glover for synthesizing the oligonucleotide primers and members of the DNA sequencing core facility for technical support. We greatly appreciate the encouragement and support from Dr. Rick Ludwig. We also thank Sandra Cockerham for helping initially some of the mammalian expression work. Results from this work were partly presented at the second

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Research reportGroup III human metabotropic glutamate receptors 4, 7 and 8: Molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgements

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Eur. J. Pharmacol. — Mol. Pharmacol. Section

Research report
Group III human metabotropic glutamate receptors 4, 7 and 8: Molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells